3-(4-amidopyrrol-2-ylmethylidene)-2-indolinone derivatives as protein kinase inhibitors

ABSTRACT

The present invention provides a method to make a pyrrole substituted 2-indolinone compound of the formula

This application is a divisional application of U.S. patent applicationSer. No. 11/511,981, filed Aug. 28, 2006, now allowed, which is adivisional application of U.S. patent application Ser. No. 10/656,907,filed Sep. 8, 2003, now U.S. Pat. No. 7,179,910, which is a divisionalapplication of U.S. patent application Ser. No. 10/076,140, filed Feb.15, 2002, now U.S. Pat. No. 6,653,308, which claims the benefit of U.S.Provisional Application Nos. 60/312,361 filed Aug. 15, 2001 and60/268,683 filed Feburary 15, 2001, the contents of which areincorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention relates to certain3-(4-amidopyrrol-2-ylmethylidene)-2-indolinone derivatives whichmodulate the activity of protein kinases (“PKs”). The compounds of thisinvention are therefore useful in treating disorders related to abnormalPK activity. Pharmaceutical compositions comprising these compounds,methods of treating diseases utilizing pharmaceutical compositionscomprising these compounds and methods of preparing them are alsodisclosed.

2. State of the Art

PKs are enzymes that catalyze the phosphorylation of hydroxy groups ontyrosine, serine and threonine residues of proteins. The consequences ofthis seemingly simple activity are staggering; cell growth,differentiation and proliferation, i.e., virtually all aspects of celllife in one way or another depend on PK activity. Furthermore, abnormalPK activity has been related to a host of disorders, ranging fromrelatively non life threatening diseases such as psoriasis to extremelyvirulent diseases such as glioblastoma (brain cancer).

The PKs can be conveniently broken down into two classes, the proteintyrosine kinases (PTKs) and the serine-threonine kinases (STKs).

One of the prime aspects of PTK activity is their involvement withgrowth factor receptors. Growth factor receptors are cell-surfaceproteins. When bound by a growth factor ligand, growth factor receptorsare converted to an active form which interacts with proteins on theinner surface of a cell membrane. This leads to phosphorylation ontyrosine residues of the receptor and other proteins and to theformation inside the cell of complexes with a variety of cytoplasmsignaling molecules that, in turn, effect numerous cellular responsessuch as cell division (proliferation), cell differentiation, cellgrowth, expression of metabolic effects to the extracellularmicroenvironment, etc. For a more complete discussion, see Schlessingerand Ullrich, Neuron, 9:303-391 (1992) which is incorporated byreference, including any drawings, as if fully set forth herein.

Growth factor receptors with PTK activity are known as receptor tyrosinekinases (“RTKs”). They comprise a large family of transmembranereceptors with diverse biological activity. At present, at leastnineteen (19) distinct subfamilies of RTKs have been identified. Anexample of these is the subfamily designated the “HER” RTKs, whichinclude EGFR (epithelial growth factor receptor), HER2, HER3 and HER4.These RTKs consist of an extracellular glycosylated ligand bindingdomain, a transmembrane domain and an intracellular cytoplasm catalyticdomain that can phosphorylate tyrosine residues on proteins.

Another RTK subfamily consists of insulin receptor (IR), insulin-likegrowth factor I receptor (IGF-1R) and insulin receptor related receptor(IRR). IR and IGF-1R interact with insulin, IGF-I and IGF-II to form aheterotetramer of two entirely extracellular glycosylated α subunits andtwo β subunits which cross the cell membrane and which contain thetyrosine kinase domain.

A third RTK subfamily is referred to as the platelet derived growthfactor receptor (“PDGFR”) group, which includes PDGFRα, PDGFRβ, CSFIR,c-kit and c-fms. These receptors consist of glycosylated extracellulardomains composed of variable numbers of immunoglobin-like loops and anintracellular domain wherein the tyrosine kinase domain is interruptedby unrelated amino acid sequences.

Another group which, because of its similarity to the PDGFR subfamily,is sometimes subsumed into the later group is the fetus liver kinase(“flk”) receptor subfamily. This group is believed to be made up ofkinase insert domain-receptor fetal liver kinase-1 (KDR/FLK-1, VEGF-R2),flk-1R, flk-4 and fms-like tyrosine kinase 1 (flt-1).

A further member of the tyrosine kinase growth factor receptor family isthe fibroblast growth factor (“FGF”) receptor subgroup. This groupconsists of four receptors, FGFR1-4, and seven ligands, FGF1-7. Whilenot yet well defined, it appears that the receptors consist of aglycosylated extracellular domain containing a variable number ofimmunoglobin-like loops and an intracellular domain in which thetyrosine kinase sequence is interrupted by regions of unrelated aminoacid sequences.

Still another member of the tyrosine kinase growth factor receptorfamily is the vascular endothelial growth factor (VEGF″) receptorsubgroup. VEGF is a dimeric glycoprotein similar to PDGF but hasdifferent biological functions and target cell specificity in vivo. Inparticular, VEGF is presently thought to play an essential role isvasculogenesis and angiogenesis.

A more complete listing of the known RTK subfamilies is described inPlowman et al., DN&P, 7(6):334-339 (1994) which is incorporated byreference, including any drawings, as if fully set forth herein.

In addition to the RTKs, there also exists a family of entirelyintracellular PTKs called “non-receptor tyrosine kinases” or “cellulartyrosine kinases.” This latter designation, abbreviated “CTK,” will beused herein. CTKs do not contain extracellular and transmembranedomains. At present, over 24 CTKs in 11 subfamilies (Src, Frk, Btk, Csk,Abl, Zap70, Fes, Fps, Fak, Jak and Ack) have been identified. The Srcsubfamily appear so far to be the largest group of CTKs and includesSrc, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. For a more detaileddiscussion of CTKs, see Bolen, Oncogene, 8:2025-2031 (1993), which isincorporated by reference, including any drawings, as if fully set forthherein.

The serine/threonine kinases, STKs, like the CTKs, are predominantlyintracellular although there are a few receptor kinases of the STK type.STKs are the most common of the cytosolic kinases; i.e., kinases thatperform their function in that part of the cytoplasm other than thecytoplasmic organelles and cytoskeleton. The cytosol is the regionwithin the cell where much of the cell's intermediary metabolic andbiosynthetic activity occurs; e.g., it is in the cytosol that proteinsare synthesized on ribosomes.

RTKs, CTKs and STKs have all been implicated in a host of pathogenicconditions including, significantly, cancer. Other pathogenic conditionswhich have been associated with PTKs include, without limitation,psoriasis, hepatic cirrhosis, diabetes, angiogenesis, restenosis, oculardiseases, rheumatoid arthritis and other inflammatory disorders,immunological disorders such as autoimmune disease, cardiovasculardisease such as atherosclerosis and a variety of renal disorders.

With regard to cancer, two of the major hypotheses advanced to explainthe excessive cellular proliferation that drives tumor developmentrelate to functions known to be PK regulated. That is, it has beensuggested that malignant cell growth results from a breakdown in themechanisms that control cell division and/or differentiation. It hasbeen shown that the protein products of a number of proto-oncogenes areinvolved in the signal transduction pathways that regulate cell growthand differentiation. These protein products of proto-oncogenes includethe extracellular growth factors, transmembrane growth factor PTKreceptors (RTKs), cytoplasmic PTKs (CTKs) and cytosolic STKs, discussedabove.

In view of the apparent link between PK-related cellular activities andwide variety of human disorders, it is no surprise that a great deal ofeffort is being expended in an attempt to identify ways to modulate PKactivity. Some of this effort has involved biomimetic approaches usinglarge molecules patterned on those involved in the actual cellularprocesses (e.g., mutant ligands (U.S. Pat. No. 4,966,849); solublereceptors and antibodies (App. No. WO 94/10202, Kendall and Thomas,Proc. Nat'l Acad. Sci., 90:10705-09 (1994), Kim, et al., Nature,362:841-844 (1993)); RNA ligands (Jelinek, et al., Biochemistry,33:10450-56); Takano, et al., Mol. Bio. Cell 4:358A (1993); Kinsella, etal., Exp. Cell Res. 199:56-62 (1992); Wright, et al., J. Cellular Phys.,152:448-57) and tyrosine kinase inhibitors (WO 94/03427; WO 92/21660; WO91/15495; WO 94/14808; U.S. Pat. No. 5,330,992; Mariani, et al., Proc.Am. Assoc. Cancer Res., 35:2268 (1994)).

In addition to the above, attempts have been made to identify smallmolecules which act as PK inhibitors. For example, bis-monocylic,bicyclic and heterocyclic aryl compounds (PCT WO 92/20642),vinyleneazaindole derivatives (PCT WO 94/14808) and1-cyclopropyl-4-pyridylquinolones (U.S. Pat. No. 5,330,992) have beendescribed as tyrosine kinase inhibitors. Styryl compounds (U.S. Pat. No.5,217,999), styryl-substituted pyridyl compounds (U.S. Pat. No.5,302,606), quinazoline derivatives (EP App. No. 0 566 266 A1),selenaindoles and selenides (PCT WO 94/03427), tricyclic polyhydroxyliccompounds (PCT WO 92/21660) and benzylphosphonic acid compounds (PCT WO91/15495) have all been described as PTK inhibitors useful in thetreatment of cancer.

SUMMARY OF THE INVENTION

The present invention is directed to certain3-(4-amidopyrrol-2-ylmethylidene)-2-indolinone derivatives which exhibitPK modulating ability and are therefore useful in treating disordersrelated to abnormal PK activity.

One embodiment of this invention is a compound of Formula (I):

wherein:

R¹ is selected from the group consisting of hydrogen, halo, alkyl,haloalkoxy, cycloalkyl, heteroalicyclic, hydroxy, alkoxy, —C(O)R⁸,—NR⁹R¹⁰ and —C(O)NR¹²R¹³;

R² is selected from the group consisting of hydrogen, halo, alkyl,trihalomethyl, hydroxy, alkoxy, cyano, —NR⁹R¹⁰, —NR⁹C(O)R¹⁰, —C(O)R⁸,—S(O)₂NR⁹R¹⁰ and —SO₂R¹⁴ (wherein R¹⁴ is alkyl, aryl, aralkyl,heteroaryl and heteroaralkyl);

R³, R⁴ and R⁵ are independently hydrogen or alkyl;

Z is aryl, heteroaryl, heterocycle, or —NR¹⁵R¹⁶ wherein R¹⁵ and R¹⁶ areindependently hydrogen or alkyl; or R¹⁵ and R¹⁶ together with thenitrogen atom to which they are attached from a heterocycloamino group;

R⁶ is selected from the group consisting of hydrogen or alkyl;

R⁷ is selected from the group consisting of hydrogen, alkyl, aryl,heteroaryl, and —C(O)R¹⁷;

R⁸ is selected from the group consisting of hydroxy, alkoxy, andaryloxy;

R⁹ and R¹⁰ are independently selected from the group consisting ofhydrogen, alkyl, cyanoalkyl, cycloalkyl, aryl and heteroaryl; or

R⁹ and R¹⁰ combine to form a heterocycloamino group;

R¹² and R¹³ are independently selected from the group consisting ofhydrogen, alkyl, hydroxyalkyl, and aryl; or R¹² and R¹³ together withthe nitrogen atom to which they are attached form a heterocycloamino;

R¹⁷ is selected from the group consisting of hydroxy, alkyl, cycloalkyl,aryl and heteroaryl;

or a pharmaceutically acceptable salt thereof.

Another embodiment is a compound of Formula I wherein

R¹ is selected from the group consisting of hydrogen, halo, alkyl,cycloalkyl, heteroalicyclic, hydroxy, alkoxy, —C(O)R⁸, —NR⁹R¹⁰ and—C(O)NR¹²R¹³;

R² is selected from the group consisting of hydrogen, halo, alkyl,trihalomethyl, hydroxy, alkoxy, cyano, —NR⁹R¹⁰, —NR⁹C(O)R¹⁰, —C(O)R⁸,—S(O)₂NR⁹R¹⁰ and —SO₂R¹⁴ (wherein R¹⁴ is alkyl, aryl, aralkyl,heteroaryl and heteroaralkyl);

R³, R⁴ and R⁵ are independently hydrogen or alkyl;

Z is aryl, heteroaryl, heterocycle, or —NR¹⁵R¹⁶ wherein R¹⁵ and R¹⁶ areindependently hydrogen or alkyl; or R¹⁵ and R¹⁶ together with thenitrogen atom to which they are attached form a heterocycloamino group;

R⁶ is selected from the group consisting of hydrogen or alkyl;

R⁷ is selected from the group consisting of hydrogen, alkyl, aryl,heteroaryl, and —C(O)R¹⁷;

R⁸ is selected from the group consisting of hydroxy, alkoxy, andaryloxy;

R⁹ and R¹⁰ are independently selected from the group consisting ofhydrogen, alkyl, cyanoalkyl, cycloalkyl, aryl and heteroaryl; or

R⁹ and R¹⁰ combine to form a heterocyclo group;

R¹² and R¹³ are independently selected from the group consisting ofhydrogen, alkyl and aryl, or R¹² and R¹³ together with the nitrogen atomto which they are attached form a heterocycle;

R¹⁷ is selected from the group consisting of hydroxy, alkyl, cycloalkyl,aryl and heteroaryl;

or a pharmaceutically acceptable salt thereof.

Another embodiment is compound of Formula (Ia):

wherein:

R¹, R³, R⁴, and R⁵ are hydrogen;

R² is fluoro and is located at the 5-position of the indolinone ring;

Z is morpholin-4-yl;

R⁶ and R⁷ are methyl.

Preferably, the stereochemistry at the *C is (S).

Another embodiment is compound of Formula (II):

wherein:

R¹ is hydrogen or alkyl;

R¹ is selected from the group consisting of hydrogen, halo, alkyl,haloalkoxy, cycloalkyl, heteroalicyclic, hydroxy, alkoxy, —C(O)R⁸,—NR⁹R¹⁰ and —C(O)NR¹²R¹³;

R² is selected from the group consisting of hydrogen, halo, alkyl,trihalomethyl, hydroxy, alkoxy, cyano, —NR⁹R¹⁰, —NR⁹C(O)R¹⁰, —C(O)R⁸,—S(O)₂NR⁹R¹⁰ and —SO₂R¹⁴ (wherein R¹⁴ is alkyl, aryl, aralkyl,heteroaryl and heteroaralkyl);

R³, R⁴ and R⁵ are independently hydrogen or alkyl;

Z is aryl, heteroaryl, heterocycle, or —NR¹⁵R¹⁶ wherein R¹⁵ and R¹⁶ areindependently hydrogen or alkyl; or R¹⁵ and R¹⁶ together with thenitrogen atom to which they are attached from a heterocycloamino group;

R⁶ is selected from the group consisting of hydrogen or alkyl;

R⁷ is selected from the group consisting of hydrogen, alkyl, aryl,heteroaryl, and —C(O)R¹⁷;

R⁸ is selected from the group consisting of hydroxy, alkoxy, andaryloxy;

R⁹ and R¹⁰ are independently selected from the group consisting ofhydrogen, alkyl, cyanoalkyl, cycloalkyl, aryl and heteroaryl; or

R⁹ and R¹⁰ combine to form a heterocycloamino group;

R¹² and R¹³ are independently selected from the group consisting ofhydrogen, alkyl, hydroxyalkyl, and aryl; or R¹² and R¹³ together withthe nitrogen atom to which they are attached form a heterocycloamino;

R¹⁷ is selected from the group consisting of hydroxy, alkyl, cycloalkyl,aryl and heteroaryl;

or a pharmaceutically acceptable salt thereof.

Another embodiment is a pharmaceutical composition, comprising acompound or salt of Formulas I, Ia, or II and a pharmaceuticallyacceptable carrier or excipient.

Another embodiment is a method for the modulation of the catalyticactivity of a protein kinase, comprising contacting the protein kinasewith a compound or salt of Formulas I, Ia, or II. The protein kinase forthis method can be a receptor tyrosine kinase, a non-receptor tyrosinekinase and a serine-threonine kinase.

Another embodiment is a method for treating or preventing a proteinkinase related disorder in an organism, comprising administering atherapeutically effective amount of a pharmaceutical compositioncomprising a compound or salt of Formulas I, Ia, or II and apharmaceutically acceptable carrier or excipient to the organism. Theprotein kinase for this method can be a receptor tyrosine kinase, anon-receptor tyrosine kinase and a serine-threonine kinase. The proteinkinase related disorder can be an EGFR related disorder, a PDGFR relateddisorder, an IGFR related disorder and a flk related disorder. Theprotein kinase disorder can also be squamous cell carcinoma,astrocytoma, Kaposi's sarcoma, glioblastoma, lung cancer, bladdercancer, head and neck cancer, melanoma, ovarian cancer, prostate cancer,breast cancer, small-cell lung cancer, glioma, colorectal cancer,genitourinary cancer and gastrointestinal cancer. Moreover, the proteinkinase disorder can also be diabetes, an autoimmune disorder, ahyperproliferation disorder, restenosis, fibrosis, psoriasis, vonHeppel-Lindau disease, osteoarthritis, rheumatoid arthritis,angiogenesis, an inflammatory disorder, an immunological disorder and acardiovascular disorder. These methods can be used to treat humans.

In another embodiment, this invention is directed to methods ofpreparing compounds of Formula (I).

Lastly, this invention is also directed to identifying a chemicalcompound that modulates the catalytic activity of a protein kinase bycontacting cells expressing the protein kinase with a compound or a saltof the present invention and then monitoring the cells for an effect.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless otherwise stated the following terms used in the specificationand claims have the meanings discussed below:

“Alkyl” refers to a saturated aliphatic hydrocarbon radical includingstraight chain and branched chain groups of 1 to 20 carbon atoms(whenever a numerical range; e.g. “1-20”, is stated herein, it meansthat the group, in this case the alkyl group, may contain 1 carbon atom,2 carbon atoms, 3 carbon atoms, etc. up to and including 20 carbonatoms). More preferably, it is a medium size alkyl having 1 to 10 carbonatoms e.g., methyl, ethyl, propyl, 2-propyl, n-butyl, iso-butyl,tert-butyl, pentyl, and the like. Most preferably, it is a lower alkylhaving 1 to 4 carbon atoms e.g., methyl, ethyl, propyl, 2-propyl,n-butyl, iso-butyl, or tert-butyl, and the like. Alkyl may besubstituted or unsubstituted, and when substituted the substituentgroup(s) is preferably halo, hydroxy, lower alkoxy, aryl, aryloxy,heteroaryl, heteroalicyclic, C(O)R⁸, NR⁹R¹⁰, and C(O)NR⁹R¹⁰.

“Cycloalkyl” refers to a 3 to 8 member all-carbon monocyclic ring, anall-carbon 5-member/6-member or 6-member/6-member fused bicyclic ring ora multicyclic fused ring (a “fused” ring system means that each ring inthe system shares an adjacent pair of carbon atoms with each other ringin the system) group wherein one or more of the rings may contain one ormore double bonds but none of the rings has a completely conjugatedpi-electron system. Examples, without limitation, of cycloalkyl groupsare cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane,cyclohexadiene, adamantane, cycloheptane, cycloheptatriene, and thelike. A cycloalkyl group may be substituted or unsubstituted. Whensubstituted, the substituent group(s) is preferably one or more, morepreferably one or two substituents, independently selected from thegroup consisting of lower alkyl, trihaloalkyl, halo, hydroxy, loweralkoxy, aryl optionally substituted with one or more, preferably one ortwo groups independently of each other halo, hydroxy, lower alkyl orlower alkoxy groups, aryloxy optionally substituted with one or more,preferably one or two groups independently of each other halo, hydroxy,lower alkyl or lower alkoxy groups, 6-member heteroaryl having from 1 to3 nitrogen atoms in the ring, the carbons in the ring being optionallysubstituted with one or more, preferably one or two groups independentlyof each other halo, hydroxy, lower alkyl or lower alkoxy groups,5-member heteroaryl having from 1 to 3 heteroatoms selected from thegroup consisting of nitrogen, oxygen and sulfur, the carbon and nitrogenatoms of the group being optionally substituted with one or more,preferably one or two groups independently of each other halo, hydroxy,lower alkyl or lower alkoxy groups, 5- or 6-member heteroalicyclic grouphaving from 1 to 3 heteroatoms selected from the group consisting ofnitrogen, oxygen and sulfur, the carbon and nitrogen (if present) atomsin the group being optionally substituted with one or more, preferablyone or two groups independently of each other halo, hydroxy, lower alkylor lower alkoxy groups, mercapto, (lower alkyl)thio, arylthio optionallysubstituted with one or more, preferably one or two groups independentlyof each other halo, hydroxy, lower alkyl or lower alkoxy groups, cyano,acyl, thioacyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, nitro, N-sulfonamido, S-sulfonamido, R⁹S(O)—,R⁹S(O)₂—, —C(O)OR⁹, R⁹C(O)O—, and —NR⁹R¹⁰ are as defined above.

“Alkenyl” refers to an alkyl group, as defined herein, consisting of atleast two carbon atoms and at least one carbon-carbon double bond.Representative examples include, but are not limited to, ethenyl,1-propenyl, 2-propenyl, 1-, 2-, or 3-butenyl, and the like.

“Alkynyl” refers to an alkyl group, as defined herein, consisting of atleast two carbon atoms and at least one carbon-carbon triple bond.Representative examples include, but are not limited to, ethynyl,1-propynyl, 2-propynyl, 1-, 2-, or 3-butynyl, and the like.

“Aryl” refers to an all-carbon monocyclic or fused-ring polycyclic(i.e., rings which share adjacent pairs of carbon atoms) groups of 1 to12 carbon atoms having a completely conjugated pi-electron system.Examples, without limitation, of aryl groups are phenyl, naphthalenyland anthracenyl. The aryl group may be substituted or unsubstituted.When substituted, the substituted group(s) is preferably one or more,more preferably one, two or three, even more preferably one or two,independently selected from the group consisting of lower alkyl,trihaloalkyl, halo, hydroxy, lower alkoxy, mercapto, (lower alkyl)thio,cyano, acyl, thioacyl, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, C-amido, N-amido, nitro, N-sulfonamido, S-sulfonamido,R⁹S(O)—, R⁹S(O)₂—, —C(O)OR⁹, R⁹C(O)O—, and —NR⁹R¹⁰, with R⁹ and R¹⁰ asdefined above. Preferably, the aryl group is optionally substituted withone or two substituents independently selected from halo, lower alkyl,trihaloalkyl, hydroxy, mercapto, cyano, N-amido, mono or dialkylamino,carboxy, or N-sulfonamido.

“Heteroaryl” refers to a monocyclic or fused ring (i.e., rings whichshare an adjacent pair of atoms) group of 5 to 12 ring atoms containingone, two, three or four ring heteroatoms selected from N, O, or S, theremaining ring atoms being C, and, in addition, having a completelyconjugated pi-electron system. Examples, without limitation, ofunsubstituted heteroaryl groups are pyrrole, furan, thiophene,imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline,isoquinoline, purine, tetrazole, triazine, and carbazole. The heteroarylgroup may be substituted or unsubstituted. When substituted, thesubstituted group(s) is preferably one or more, more preferably one,two, or three, even more preferably one or two, independently selectedfrom the group consisting of lower alkyl, trihaloalkyl, halo, hydroxy,lower alkoxy, mercapto, (lower alkyl)thio, cyano, acyl, thioacyl,O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,N-amido, nitro, N-sulfonamido, S-sulfonamido, R⁹S(O)—, R⁹O)₂—, —C(O)OR⁹,R⁹C(O)O—, and —NR⁹R¹⁰, with R⁹ and R¹⁰ as defined above. Preferably, theheteroaryl group is optionally substituted with one or two substituentsindependently selected from halo, lower alkyl, trihaloalkyl, hydroxy,mercapto, cyano, N-amido, mono or dialkylamino, carboxy, orN-sulfonamido.

“Heteroalicyclic” refers to a monocyclic or fused ring group having inthe ring(s) of 5 to 9 ring atoms in which one or two ring atoms areheteroatoms selected from N, O, or S(O)_(n) (where n is an integer from0 to 2), the remaining ring atoms being C. The rings may also have oneor more double bonds. However, the rings do not have a completelyconjugated pi-electron system. Examples, without limitation, ofunsubstituted heteroalicyclic groups are pyrrolidino, piperidino,piperazino, morpholino, thiomorpholino, homopiperazino, and the like.The heteroalicyclic ring may be substituted or unsubstituted. Whensubstituted, the substituted group(s) is preferably one or more, morepreferably one, two or three, even more preferably one or two,independently selected from the group consisting of lower alkyl,trihaloalkyl, halo, hydroxy, lower alkoxy, mercapto, (lower alkyl)thio,cyano, acyl, thioacyl, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, C-amido, N-amido, nitro, N-sulfonamido, S-sulfonamido,R⁹S(O)—, R⁹S(O)₂—, —C(O)OR⁹, R⁹C(O)O—, and —NR⁹R¹⁰, with R⁹ and R¹⁰ asdefined above. Preferably, the heteroalicyclic group is optionallysubstituted with one or two substituents independently selected fromhalo, lower alkyl, trihaloalkyl, hydroxy, mercapto, cyano, N-amido, monoor dialkylamino, carboxy, or N-sulfonamido.

“Heterocycle” means a saturated cyclic radical of 3 to 8 ring atoms inwhich one or two ring atoms are heteroatoms selected from N, O, orS(O)_(n) (where n is an integer from 0 to 2), the remaining ring atomsbeing C, where one or two C atoms may optionally be replaced by acarbonyl group. The heterocyclyl ring may be optionally substitutedindependently with one, two, or three substituents selected from loweralkyl optionally substituted one or two substituents independentlyselected from carboxy or ester group, haloalkyl, cyanoalkyl, halo,nitro, cyano, hydroxy, alkoxy, amino, monoalkylamino, dialkylamino,aralkyl, heteroaralkyl, and —COR (where R is alkyl). More specificallythe term heterocyclyl includes, but is not limited to,tetrahydropyranyl, 2,2-dimethyl-1,3-dioxolane, piperidino,N-methylpiperidin-3-yl, piperazino, N-methylpyrrolidin-3-yl,pyrrolidino, morpholino, thiomorpholino, thiomorpholino-1-oxide,thiomorpholino-1,1-dioxide, 4-ethyloxycarbonypiperazino,3-oxopiperazino, 2-imidazolidone, 2-pyrrolidinone, 2-oxohomopiperazino,tetrahydropyrimidin-2-one, and the derivatives thereof. Preferably, theheterocycle group is optionally substituted with one or two substituentsindependently selected from halo, lower alkyl, lower alkyl substitutedwith carboxy, ester hydroxy, or mono or dialkylamino.

“Heterocycloamino” means a saturated cyclic radical of 3 to 8 ring atomsin which at least one of the ring atoms is nitrogen and optionally whereone or two additionally ring atoms are heteroatoms selected from N, O,or S(O)_(n) (where n is an integer from 0 to 2), the remaining ringatoms being C, where one or two C atoms may optionally be replaced by acarbonyl group. The heterocycloamino ring may be optionally substitutedindependently with one, two, or three substituents selected from loweralkyl optionally substituted one or two substituents independentlyselected from carboxy or ester group, haloalkyl, cyanoalkyl, halo,nitro, cyano, hydroxy, alkoxy, amino, monoalkylamino, dialkylamino,aralkyl, heteroaralkyl, and —COR (where R is alkyl. More specificallythe term heterocycloamino includes, but is not limited to, piperidin1-yl, piperazin-1-yl, pyrrolidin-1-yl, morpholin-4-yl,thiomorpholin-4-yl, thiomorpholino-1-oxide, thiomorpholino-1,1-dioxide,4-ethyloxycarbonylpiperazin-1-yl, 3-oxopiperazin-1-yl,2-imidazolidon-1-yl, 2-pyrrolidinon-1-yl, 2-oxohomopiperazino,tetrahydropyrimidin-2-one, and the derivatives thereof. Preferably, theheterocycle group is optionally substituted with one or two substituentsindependently selected from halo, lower alkyl, lower alkyl substitutedwith carboxy or ester, hydroxy, or mono or dialkylamino. Theheterocycloamino group is a subset of the heterocycle group definedabove.

“Hydroxy” refers to an —OH group.

“Alkoxy” refers to both an —O-(alkyl) and an —O-(unsubstitutedcycloalkyl) group. Representative examples include, but are not limitedto, e.g., methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy,cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like.

“Haloalkoxy” refers to both an —O-(haloalkyl) group. Representativeexamples include, but are not limited to, e.g., trifluoromethoxy,tribromomethoxy, and the like.

“Aryloxy” refers to both an —O-aryl and an —O-heteroaryl group, asdefined herein. Representative examples include, but are not limited to,phenoxy, pyridinyloxy, furanyloxy, thienyloxy, pyrimidinyloxy,pyrazinyloxy, and the like, and derivatives thereof.

“Mercapto” refers to an —SH group.

“Alkylthio” refers to both an —S-(alkyl) and an —S-(unsubstitutedcycloalkyl) group. Representative examples include, but are not limitedto, e.g., methylthio, ethylthio, propylthio, butylthio, cyclopropylthio,cyclobutylthio, cyclopentylthio, cyclohexylthio, and the like.

“Arylthio” refers to both an —S-aryl and an —S-heteroaryl group, asdefined herein. Representative examples include, but are not limited to,phenylthio, pyridinylthio, furanylthio, thienylthio, pyrimidinylthio,and the like and derivatives thereof.

“Acyl” refers to a —C(O)—R″ group, where R″ is selected from the groupconsisting of hydrogen, lower alkyl, trihalomethyl, unsubstitutedcycloalkyl, aryl optionally substituted with one or more, preferablyone, two, or three substituents selected from the group consisting oflower alkyl, trihalomethyl, lower alkoxy, halo and —NR⁹R¹⁰ groups,heteroaryl (bonded through a ring carbon) optionally substituted withone or more, preferably one, two, or three substitutents selected fromthe group consisting of lower alkyl, trihaloalkyl, lower alkoxy, haloand —NR⁹R¹⁰ groups and heteroalicyclic (bonded through a ring carbon)optionally substituted with one or more, preferably one, two, or threesubstituents selected from the group consisting of lower alkyl,trihaloalkyl, lower alkoxy, halo and —NR⁹R¹⁰ groups. Representative acylgroups include, but are not limited to, acetyl, trifluoroacetyl,benzoyl, and the like

“Aldehyde” refers to an acyl group in which R″ is hydrogen.

“Thioacyl” refers to a —C(S)—R″ group, with R″ as defined herein.

“Ester” refers to a —C(O)O—R″ group with R″ as defined herein exceptthat R″ cannot be hydrogen.

“Acetyl” group refers to a —C(O)CH₃ group.

“Halo” group refers to fluorine, chlorine, bromine or iodine, preferablyfluorine or chlorine.

“Trihalomethyl” group refers to a —CX₃ group wherein X is a halo asdefined above.

“Trihalomethanesulfonyl” group refers to a X₃CS(═O)₂— groups with X asdefined above.

“Cyano” refers to a —C≡N group.

“S-sulfonamido” refers to a —S(O)₂NR⁹R¹⁰ group, with R⁹ and R¹⁰ asdefined herein.

“N-sulfonamido” refers to a —NR⁹S(O)₂R¹⁰ group, with R⁹ and R¹⁰ asdefined herein.

“O-carbamyl” group refers to a —OC(O)NR¹²R¹³ group with R¹² and R¹³ asdefined herein.

“N-carbamyl” refers to an R⁹OC(O)NR¹⁰— group, with R⁹ and R¹⁰ as definedherein.

“O-thiocarbamyl” refers to a —OC(S)NR¹²R¹³ group with R¹² and R¹³ asdefined herein.

“N-thiocarbamyl” refers to a R⁹OC(S)NR¹⁰— group, with R⁹ and R¹⁰ asdefined herein.

“Amino” refers to an —NR⁹R¹⁰ group, wherein R⁹ and R¹⁰ are bothhydrogen.

“C-amido” refers to a —C(O)NR⁹R¹⁰ group with R⁹ and R¹⁰ as definedherein.

“N-amido” refers to a R⁹C(O)NR¹⁰— group, with R⁹ and R¹⁰ as definedherein.

“Nitro” refers to a —NO₂ group.

“Haloalkyl” means an alkyl, preferably lower alkyl as defined above thatis substituted with one or more same or different halo atoms, e.g.,—CH₂Cl, —CF₃, —CH₂CF₃, —CH₂CCl₃, and the like.

“Hydroxyalkyl” means an alkyl, preferably lower alkyl as defined abovethat is substituted with one, two, or three hydroxy groups, e.g.,hydroxymethyl, 1 or 2-hydroxyethyl, 1,2-, 1,3-, or 2,3-dihydroxypropyl,and the like.

“Aralkyl” means alkyl, preferably lower alkyl as defined above which issubstituted with an aryl group as defined above, e.g., —CH₂phenyl,—(CH₂)₂phenyl, —(CH₂)₃phenyl, CH₃CH(CH₃)CH₂phenyl, and the like andderivatives thereof.

“Heteroaralkyl” group means alkyl, preferably lower alkyl as definedabove which is substituted with a heteroaryl group, e.g., —CH₂pyridinyl,—(CH₂)₂pyrimidinyl, —(CH₂)₃imidazolyl, and the like, and derivativesthereof.

“Monoalkylamino” means a radical —NHR where R is an alkyl orunsubstituted cycloalkyl group as defined above, e.g., methylamino,(1-methylethyl)amino, cyclohexylamino, and the like.

“Dialkylamino” means a radical —NRR where each R is independently analkyl or unsubstituted cycloalkyl group as defined above, e.g.,dimethylamino, diethylamino, (1-methylethyl)-ethylamino,cyclohexylmethylamino, cyclopentylmethylamino, and the like.

“Optional” or “optionally” means that the subsequently described eventor circumstance may but need not occur, and that the descriptionincludes instances where the event or circumstance occurs and instancesin which it does not. For example, “heterocycle group optionallysubstituted with an alkyl group” means that the alkyl may but need notbe present, and the description includes situations where theheterocycle group is substituted with an alkyl group and situationswhere the heterocyclo group is not substituted with the alkyl group.

The terms “2-indolinone”, “indolin-2-one” and “2-oxindole” are usedinterchangeably herein to refer to a molecule having the chemicalstructure:

The term “pyrrole” refers to a molecule having the chemical structure:

Compounds that have the same molecular formula but differ in the natureor sequence of bonding of their atoms or the arrangement of their atomsin space are termed “isomers”. Isomers that differ in the arrangement oftheir atoms in space are termed “stereoisomers”. Stereoisomers that arenot mirror images of one another are termed “diastereomers” and thosethat are non-superimposable mirror images of each other are termed“enantiomers”. When a compound has an asymmetric center, for example, itis bonded to four different groups, a pair of enantiomers is possible.An enantiomer can be characterized by the absolute configuration of itsasymmetric center and is described by the R- and S-sequencing rules ofCahn and Prelog, or by the manner in which the molecule rotates theplane of polarized light and designated as dextrorotatory orlevorotatory (i.e., as (+) or (−)-isomers respectively). A chiralcompound can exist as either individual enantiomer or as a mixturethereof. A mixture containing equal proportions of the enantiomers iscalled a “racemic mixture”.

The compounds of this invention may possess one or more asymmetriccenters; such compounds can therefore be produced as individual (R)- or(S)-stereoisomers or as mixtures thereof. For example, the carbon atomcarrying the hydroxy group in —CONHCHR³—CR⁴(OH)CR⁵Z in a compound offormula (I) is an asymmetric center and therefore the compound ofFormula (I) can exist as an (R)- or (S)-stereoisomer. Unless indicatedotherwise, the description or naming of a particular compound in thespecification and claims is intended to include both individualenantiomers and mixtures, racemic or otherwise, thereof. The methods forthe determination of stereochemistry and the separation of stereoisomersare well-known in the art (see discussion in Chapter 4 of “AdvancedOrganic Chemistry”, 4th edition J. March, John Wiley and Sons, New York,1992).

The compounds of Formula (I) may exhibit the phenomena of tautomerismand structural isomerism. For example, the compounds described hereinmay adopt an E or a Z configuration about the double bond connecting the2-indolinone moiety to the pyrrole moiety or they may be a mixture of Eand Z. This invention encompasses any tautomeric or structural isomericform and mixtures thereof which possess the ability to modulate RTK, CTKand/or STK activity and is not limited to any one tautomeric orstructural isomeric form.

A “pharmaceutical composition” refers to a mixture of one or more of thecompounds described herein, or physiologically/pharmaceuticallyacceptable salts or prodrugs thereof, with other chemical components,such as physiologically/pharmaceutically acceptable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

The compound of Formula (I) may also act as a prodrug. A “prodrug”refers to an agent which is converted into the parent drug in vivo.Prodrugs are often useful because, in some situations, they may beeasier to administer than the parent drug. They may, for instance, bebioavailable by oral administration whereas the parent drug is not. Theprodrug may also have improved solubility in pharmaceutical compositionsover the parent drug. An example, without limitation, of a prodrug wouldbe a compound of the present invention which is administered as an ester(the “prodrug”) to facilitate transmittal across a cell membrane wherewater solubility is detrimental to mobility but then is metabolicallyhydrolyzed to the carboxylic acid, the active entity, once inside thecell where water solubility is beneficial.

A further example of a prodrug might be a short polypeptide, forexample, without limitation, a 2-10 amino acid polypeptide, bondedthrough a terminal amino group to a carboxy group of a compound of thisinvention wherein the polypeptide is hydrolyzed or metabolized in vivoto release the active molecule. The prodrugs of a compound of Formula(I) are within the scope of this invention.

Additionally, it is contemplated that a compound of Formula (I) would bemetabolized by enzymes in the body of the organism such as a human beingto generate a metabolite that can modulate the activity of the proteinkinases. Such metabolites are within the scope of the present invention.

As used herein, a “physiologically/pharmaceutically acceptable carrier”refers to a carrier or diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound.

An “pharmaceutically acceptable excipient” refers to an inert substanceadded to a pharmaceutical composition to further facilitateadministration of a compound. Examples, without limitation, ofexcipients include calcium carbonate, calcium phosphate, various sugarsand types of starch, cellulose derivatives, gelatin, vegetable oils andpolyethylene glycols.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which retain the biological effectiveness and properties ofthe parent compound. Such salts include:

(i) acid addition salt which is obtained by reaction of the free base ofthe parent compound with inorganic acids such as hydrochloric acid,hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, andperchloric acid and the like, or with organic acids such as acetic acid,oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaricacid, citric acid, succinic acid or malonic acid and the like,preferably hydrochloric acid or (L)-malic; or

(2) salts formed when an acidic proton present in the parent compoundeither is replaced by a metal ion, e.g., an alkali metal ion, analkaline earth ion, or an aluminum ion; or coordinates with an organicbase such as ethanolamine, diethanolamine, triethanolamine,tromethamine, N-methylglucamine, and the like.

“PK” refers to receptor protein tyrosine kinase (RTKs), non-receptor or“cellular” tyrosine kinase (CTKs) and serine-threonine kinases (STKs).

“Method” refers to manners, means, techniques and procedures foraccomplishing a given task including, but not limited to, those manners,means, techniques and procedures either known to, or readily developedfrom known manners, means, techniques and procedures by, practitionersof the chemical, pharmaceutical, biological, biochemical and medicalarts.

“Modulation” or “modulating” refers to the alteration of the catalyticactivity of RTKs, CTKs and STKs. In particular, modulating refers to theactivation of the catalytic activity of RTKs, CTKs and STKs, preferablythe activation or inhibition of the catalytic activity of RTKs, CTKs andSTKs, depending on the concentration of the compound or salt to whichthe RTK, CTK or STK is exposed or, more preferably, the inhibition ofthe catalytic activity of RTKs, CTKs and STKs.

“Catalytic activity” refers to the rate of phosphorylation of tyrosineunder the influence, direct or indirect, of RTKs and/or CTKs or thephosphorylation of serine and threonine under the influence, direct orindirect, of STKs.

“Contacting” refers to bringing a compound of this invention and atarget PK together in such a manner that the compound can affect thecatalytic activity of the PK, either directly, i.e., by interacting withthe kinase itself, or indirectly, i.e., by interacting with anothermolecule on which the catalytic activity of the kinase is dependent.Such “contacting” can be accomplished “in vitro,” i.e., in a test tube,a petri dish or the like. In a test tube, contacting may involve only acompound and a PK of interest or it may involve whole cells. Cells mayalso be maintained or grown in cell culture dishes and contacted with acompound in that environment. In this context, the ability of aparticular compound to affect a PK related disorder, i.e., the IC₅₀ ofthe compound, defined below, can be determined before use of thecompounds in vivo with more complex living organisms is attempted. Forcells outside the organism, multiple methods exist, and are well-knownto those skilled in the art, to get the PKs in contact with thecompounds including, but not limited to, direct cell microinjection andnumerous transmembrane carrier techniques.

“In vitro” refers to procedures performed in an artificial environmentsuch as, e.g., without limitation, in a test tube or culture medium.

“In vivo” refers to procedures performed within a living organism suchas, without limitation, a mouse, rat or rabbit.

“PK related disorder,” “PK driven disorder,” and “abnormal PK activity”all refer to a condition characterized by inappropriate, i.e., under or,more commonly, over, PK catalytic activity, where the particular PK canbe an RTK, a CTK or an STK. Inappropriate catalytic activity can ariseas the result of either: (1) PK expression in cells which normally donot express PKs, (2) increased PK expression leading to unwanted cellproliferation, differentiation and/or growth, or, (3) decreased PKexpression leading to unwanted reductions in cell proliferation,differentiation and/or growth. Over-activity of a PK refers to eitheramplification of the gene encoding a particular PK or production of alevel of PK activity which can correlate with a cell proliferation,differentiation and/or growth disorder (that is, as the level of the PKincreases, the severity of one or more of the symptoms of the cellulardisorder increases). Under-activity is, of course, the converse, whereinthe severity of one or more symptoms of a cellular disorder increase asthe level of the PK activity decreases.

“Treat”, “treating” and “treatment” refer to a method of alleviating orabrogating a PK mediated cellular disorder and/or its attendantsymptoms. With regard particularly to cancer, these terms simply meanthat the life expectancy of an individual affected with a cancer will beincreased or that one or more of the symptoms of the disease will bereduced.

“Organism” refers to any living entity comprised of at least one cell. Aliving organism can be as simple as, for example, a single eukarioticcell or as complex as a mammal, including a human being.

“Therapeutically effective amount” refers to that amount of the compoundbeing administered which will relieve to some extent one or more of thesymptoms of the disorder being treated. In reference to the treatment ofcancer, a therapeutically effective amount refers to that amount whichhas the effect of:

-   -   (1) reducing the size of the tumor;    -   (2) inhibiting (that is, slowing to some extent, preferably        stopping) tumor metastasis;    -   (3) inhibiting to some extent (that is, slowing to some extent,        preferably stopping) tumor growth, and/or,    -   (4) relieving to some extent (or, preferably, eliminating) one        or more symptoms associated with the cancer.

“Monitoring” means observing or detecting the effect of contacting acompound with a cell expressing a particular PK. The observed ordetected effect can be a change in cell phenotype, in the catalyticactivity of a PK or a change in the interaction of a PK with a naturalbinding partner. Techniques for observing or detecting such effects arewell-known in the art.

The above-referenced effect is selected from a change or an absence ofchange in a cell phenotype, a change or absence of change in thecatalytic activity of said protein kinase or a change or absence ofchange in the interaction of said protein kinase with a natural bindingpartner in a final aspect of this invention.

“Cell phenotype” refers to the outward appearance of a cell or tissue orthe biological function of the cell or tissue. Examples, withoutlimitation, of a cell phenotype are cell size, cell growth, cellproliferation, cell differentiation, cell survival, apoptosis, andnutrient uptake and use. Such phenotypic characteristics are measurableby techniques well-known in the art.

“Natural binding partner” refers to a polypeptide that binds to aparticular PK in a cell. Natural binding partners can play a role inpropagating a signal in a PK-mediated signal transduction process. Achange in the interaction of the natural binding partner with the PK canmanifest itself as an increased or decreased concentration of thePK/natural binding partner complex and, as a result, in an observablechange in the ability of the PK to mediate signal transduction.

Representative compounds of the present invention are shown in Table 1abelow.

TABLE 1a Cpd No. Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

47

48

49

50

51

52

Cpd No. Name MS m/z 1 5-(5-Fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(3-diethylamino-2-hydroxy- propyl)-amide 427 [M + 1] 25-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(2-hydroxy-3-morpholin-4- yl-propyl)-amide 441 [M − 1] 32,4-Dimethyl-5-[2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-1H-pyrrole-3- carboxylic acid(2-hydroxy-3-morpholin-4- yl-propyl)-amide 423 [M − 1] 45-[5-Chloro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(2-hydroxy-3-morpholin-4- yl-propyl)-amide 457 [M − 1] 55-[5-Bromo-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(2-hydroxy-3-morpholin-4- yl-propyl)-amide 501 [M − 1] 503 [M − 1] 62,4-Dimethyl-5-[2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-1H-pyrrole-3- carboxylic acid(2-hydroxy-3-[1,2,3]triazol- 1-yl-propyl)-amide 405 [M − 1] 75-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(2-hydroxy-3-[1,2,3]triazol- 1-yl-propyl)-amide 423 [M − 1] 85-[5-Chloro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(2-hydroxy-3-[1,2,3]triazol- 1-yl-propyl)-amide 439 [M − 1] 95-[5-Bromo-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(2-hydroxy-3-[1,2,3]triazol- 1-yl-propyl)-amide 483 [M − 1] 485 [M − 1]10 5-{(Z)-[4-(3-chlorophenyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]methyl}-N-(2-hydroxy-3-pyrrolidin-1-ylpropyl)-2,4-dimethyl-1H- pyrrole-3-carboxamide 11(3Z)-3-{[4-({[3-(diethylamino)-2-hydroxypropyl]amino}carbonyl)-5-methyl-3-phenyl-1H-pyrrol-2-yl]methylene}-2-oxo-N-phenyl-2,3-dihydro-1H-indole-5- carboxamide 12(3Z)-3-{[4-({[3-(diethylamino)-2-hydroxypropyl]amino}carbonyl)-5-methyl-3-phenyl-1H-pyrrol-2-yl]methylene}-N-methyl-2-oxo-2,3-dihydro-1H-indole-5-carboxamide 13(3Z)-3-{[4-({[3-(diethylamino)-2-hydroxypropyl]amino}carbonyl)-5-methyl-3-phenyl-1H-pyrrol-2-yl]methylene}-N-(2-hydroxyethyl)-2-oxo-2,3-dihydro-1H-indole-5- carboxamide 14N-[3-(diethylamino)-2-hydroxypropyl]-4-(4-fluorophenyl)-2-methyl-5-{(Z)-[5-(morpholin-4-ylcarbonyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]methyl}-1H-pyrrole-3-carboxamide 15(3Z)-3-{[4-({[3-(diethylamino)-2- hydroxypropyl]amino}carbonyl)-3-(4-fluorophenyl)-5-methyl-1H-pyrrol-2-yl]methylene}-N-isopropyl-2-oxo-2,3-dihydro- 1H-indole-5-carboxamide 16(3Z)-3-{[4-({[3-(diethylamino)-2- hydroxypropyl]amino}carbonyl)-3-(2,4-difluorophenyl)-5-methyl-1H-pyrrol-2-yl]methylene}-2-oxo-N-phenyl-2,3-dihydro- 1H-indole-5-carboxamide 17(3Z)-3-{[4-({[3-(diethylamino)-2- hydroxypropyl]amino}carbonyl)-3-(2,4-difluorophenyl)-5-methyl-1H-pyrrol-2-yl]methylene}-N-(2-hydroxyethyl)-2-oxo-2,3-dihydro-1H-indole-5-carboxamide 18 (3Z)-3-{[3-(4-cyanophenyl)-4-({[3-(diethylamino)-2- hydroxypropyl]amino}carbonyl)-5-methyl-1H-pyrrol-2-yl]methylene}-N,N-dimethyl-2-oxo-2,3-dihydro-1H-indole-5-carboxamide 194-(4-cyanophenyl)-N-[3-(diethylamino)-2-hydroxypropyl]-2-methyl-5-{(Z)-[5-(morpholin-4-ylcarbonyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]methyl}-1H-pyrrole-3- carboxamide 20(3Z)-3-{[3-(4-chlorophenyl)-4-({[3- (diethylamino)-2-hydroxypropyl]amino}carbonyl)-5-methyl-1H-pyrrol-2-yl]methylene}-2-oxo-N-phenyl-2,3-dihydro-1H-indole-5-carboxamide 21 (3Z)-3-{[3-(4-chlorophenyl)-4-({[3-(diethylamino)-2- hydroxypropyl]amino}carbonyl)-5-methyl-1H-pyrrol-2-yl]methylene}-N-isopropyl-2-oxo-2,3-dihydro-1H-indole-5-carboxamide 225-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[2-hydroxy-3-(2H-tetraazol-2-yl)propyl]-2,4-dimethyl-1H- pyrrole-3-carboxamide 235-[(Z)-(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[2-hydroxy-3-(2H-tetraazol-2-yl)propyl]-2,4-dimethyl-1H- pyrrole-3-carboxamide 24N-[2-hydroxy-3-(2H-tetraazol-2-yl)propyl]- 2,4-dimethyl-5-{(Z)-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]methyl}-1H-pyrrole-3-carboxamide 255-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[2-hydroxy-3-(1H-tetraazol-1-yl)propyl]-2,4-dimethyl-1H- pyrrole-3-carboxamide 265-[(Z)-(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[2-hydroxy-3-(1H-tetraazol-1-yl)propyl]-2,4-dimethyl-1H- pyrrole-3-carboxamide 27N-[2-hydroxy-3-(1H-tetraazol-1-yl)propyl]- 2,4-dimethyl-5-{(Z)-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]methyl}-1H-pyrrole-3-carboxamide 28N-{3-[(2R,6S)-2,6-dimethylmorpholin-4-yl]-2-hydroxypropyl}-5-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide 295-[(Z)-(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-{3-[(2R,6S)-2,6-dimethylmorpholin-4-yl]-2-hydroxypropyl}-2,4-dimethyl-1H-pyrrole-3-carboxamide 30N-{3-[(2R,6S)-2,6-dimethylmorpholin-4-yl]-2-hydroxypropyl}-2,4-dimethyl-5-{(Z)-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]methyl}-1H-pyrrole-3-carboxamide 315-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[(2R)-2-hydroxy-3-(3-methyl-2,5-dioxoimidazolidin-1-yl)propyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide 325-[(Z)-(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[(2R)-2-hydroxy-3-(3-methyl-2,5-dioxoimidazolidin-1-yl)propyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide 33N-[(2R)-2-hydroxy-3-(3-methyl-2,5-dioxoimidazolidin-1-yl)propyl]-2,4-dimethyl-5-{(Z)-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]methyl}-1H-pyrrole-3- carboxamide 345-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[(2S)-2-hydroxy-3-(3-methyl-2,5-dioxoimidazolidin-1-yl)propyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide 35N-[(2S)-2-hydroxy-3-(3-methyl-2,5-dioxoimidazolidin-1-yl)propyl]-2,4-dimethyl-5-{(Z)-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]methyl}-1H-pyrrole-3- carboxamide 365-[(Z)-(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[(2S)-2-hydroxy-3-(3-methyl-2,5-dioxoimidazolidin-1-yl)propyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide 37N-[3-(1,1-dioxidothiomorpholin-4-yl)-2-hydroxypropyl]-2,4-dimethyl-5-[(Z)-(2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-1H- pyrrole-3-carboxamide 38N-[3-(1,1-dioxidothiomorpholin-4-yl)-2-hydroxypropyl]-5-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide 395-[(Z)-(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[3-(1,1-dioxidothiomorpholin-4-yl)-2-hydroxypropyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide 405-[(Z)-(5-bromo-2-oxo-1,2-dihydro-3H-indol- 3-ylidene)methyl]-N-[3-(1,1-dioxidothiomorpholin-4-yl)-2-hydroxypropyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide 41 442.495-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[(2S)-2-hydroxy-3-morpholin-4-ylpropyl]-2,4-dimethyl-1H-pyrrole- 3-carboxamide 42 442.495-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[(2R)-2-hydroxy-3-morpholin-4-ylpropyl]-2,4-dimethyl-1H-pyrrole- 3-carboxamide 43 458.955-[(Z)-(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[(2R)-2-hydroxy-3-morpholin-4-ylpropyl]-2,4-dimethyl-1H-pyrrole- 3-carboxamide 44 458.955-[(Z)-(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[(2S)-2-hydroxy-3-morpholin-4-ylpropyl]-2,4-dimethyl-1H-pyrrole- 3-carboxamide 475-(5-(Z)-fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H- pyrrole-3-carboxylic acid [2-hydroxy-3-([1,2,3]triazolo[4,5-b]pyridin- 3-yloxy)-propyl]-amide 485-(5-(Z)-chloro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H- pyrrole-3-carboxylic acid [2-hydroxy-3-([1,2,3]triazolo[4,5-b]pyridin- 3-yloxy)-propyl]-amide 492,4-(Z)-dimethyl-5-(2-oxo-5- trifluoromethoxy-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-3-carboxylic acid[2-hydroxy-3-([1,2,3]triazolo[4,5- b]pyridin-3-yloxy)-propyl]-amide 505-(5-(Z)-fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H- pyrrole-3-carboxylic acid [2-hydroxy-3-(3-oxy-benzotriazol-1-yl)- propyl]-amide 515-(5-(Z)-chloro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H- pyrrole-3-carboxylic acid [2-hydroxy-3-(3-oxy-benzotriazol-1-yl)- propyl]-amide 522,4-(Z)-dimethyl-5-(2-oxo-5- trifluoromethoxy-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-3-carboxylic acid[2-hydroxy-3-(3-oxy-benzotriazol- 1-yl)-propyl]-amide

Other representative compounds of the present invention are shown inTable 1b below.

TABLE 1b Cpd No. Structure Name 1N

5-(5-Fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(3-diethylamino-2-hydroxy- propyl)-amide 2N

5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(2-hydroxy-3-morpholin-4-yl- propyl)-amide 3N

2,4-Dimethyl-5-[2-oxo-1,2-dihydro-indol-(3)-ylidenemethyl]-1H-pyrrole-3-carboxylic acid(2-hydroxy-3-morpholin-4-yl-propyl)-amide 4N

5-[5-Chloro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(2-hydroxy-3-morpholin-4-yl- propyl)-amide 5N

5-[5-Bromo-2-oxo-1,2-dihydro-indol-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(2-hydroxy-3-morpholin-4-yl- propyl)-amide 6N

2,4-Dimethyl-5-[2-oxo-1,2-dihydro-indol-ylidenemethyl]-1H-pyrrole-3-carboxylic acid(2-hydroxy-3-[1,2,3]triazol-1-yl-propyl)-amide 7N

5-[5-Fluoro-2-oxo-1,2-dihydro-indol-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(2-hydroxy-3-[1,2,3]triazol-1- yl-propyl)-amide 8N

5-[5-Chloro-2-oxo-1,2-dihydro-indol-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(2-hydroxy-3-[1,2,3]triazol-1- yl-propyl)-amide 9N

5-[5-Bromo-2-oxo-1,2-dihydro-indol-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(2-hydroxy-3-[1,2,3]triazol-1- yl-propyl)-amide 11N

3-{[4-({[3-(diethylamino)-2- hydroxypropyl]amino}carbonyl)-5-methyl-3-phenyl-1H-pyrrol-2-yl]methylene}-2-oxo-N-phenyl-2,3-dihydro-1H-indole-5-carboxamide 12N

(3-{[4-({[3-(diethylamino)-2- hydroxypropyl]amino}carbonyl)-5-methyl-3-phenyl-1H-pyrrol-2-yl]methylene}-N-methyl-2-oxo-2,3-dihydro-1H-indole-5-carboxamide 13N

(3Z)-3-{[4-({[3-(diethylamino)-2-hydroxypropyl]amino}carbonyl)-5-methyl-3-phenyl-1H-pyrrol-2-yl]methylene}-N-(2-hydroxyethyl)-2-oxo-2,3-dihydro-1H-indole-5- carboxamide 14N

N-[3-(diethylamino)-2-hydroxypropyl]-4-(4-fluorophenyl)-2-methyl-5-{[5-(morpholin-4-ylcarbonyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]methyl}-1H-pyrrole-3-carboxamide 15N

3-{[4-({[3-(diethylamino)-2- hydroxypropyl]amino}carbonyl)-3-(4-fluorophenyl)-5-methyl-1H-pyrrol-2- yl]methylene}-N-isopropyl-2-oxo-2,3-dihydro-1H-indole-5-carboxamide 16N

3-{[4-({[3-(diethylamino)-2- hydroxypropyl]amino}carbonyl)-3-(2,4-difluorophenyl)-5-methyl-1H-pyrrol-2-yl]methylene}-2-oxo-N-phenyl-2,3-dihydro- 1H-indole-5-carboxamide 17N

3-{[4-({[3-(diethylamino)-2- hydroxypropyl]amino}carbonyl)-3-(2,4-difluorophenyl)-5-methyl-1H-pyrrol-2-yl]methylene}-N-(2-hydroxyethyl)-2-oxo-2,3-dihydro-1H-indole-5-carboxamide 18N

3-{[3-(4-cyanophenyl)-4-({[3-(diethylamino)-2-hydroxypropyl]amino}carbonyl)-5-methyl-1H-pyrrol-2-yl]methylene}-N,N-dimethyl-2-oxo-2,3-dihydro-1H-indole-5-carboxamide 19N

4-(4-cyanophenyl)-N-[3-(diethylamino)-2-hydroxypropyl]-2-methyl-5-{[5-(morpholin-4-ylcarbonyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]methyl}-1H-pyrrole-3-carboxamide 20N

3-{[3-(4-chlorophenyl)-4-({[3-(diethylamino)-2-hydroxypropyl]amino}carbonyl)-5-methyl-1H-pyrrol-2-yl]methylene}-2-oxo-N-phenyl-2,3-dihydro-1H-indole-5-carboxamide 21N

3-{[3-(4-chlorophenyl)-4-({[3-(diethylamino)-2-hydroxypropyl]amino}carbonyl)-5-methyl-1H-pyrrol-2-yl]methylene}-N-isopropyl-2-oxo-2,3-dihydro-1H-indole-5-carboxamide 22N

5-[(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[2-hydroxy-3-(2H-tetraazol-2-yl)propyl]-2,4-dimethyl-1H-pyrrole-3- carboxamide 23N

5-[5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[2-hydroxy-3-(2H-tetraazol-2-yl)propyl]-2,4-dimethyl-1H-pyrrole-3- carboxamide 24N

N-[2-hydroxy-3-(2H-tetraazol-2-yl)propyl]-2,4-dimethyl-5-{[2-oxo-5-trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]methyl}-1H- pyrrole-3-carboxamide 25N

5-[(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[2-hydroxy-3-(1H-tetraazol-1-yl)propyl]-2,4-dimethyl-1H-pyrrole-3- carboxamide 26N

5-[(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[2-hydroxy-3-(1H-tetraazol-1-yl)propyl]-2,4-dimethyl-1H-pyrrole-3- carboxamide 27N

N-[2-hydroxy-3-(1H-tetraazol-1-yl)propyl]-2,4-dimethyl-5-{[2-oxo-5-trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]methyl}-1H- pyrrole-3-carboxamide 28N

N-{3-[2,6-dimethylmorpholin-4-yl]-2-hydroxypropyl}-5-[(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide 29N

5-[(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-{3-[2,6-dimethylmorpholin-4-yl]-2-hydroxypropyl}-2,4-dimethyl-1H- pyrrole-3-carboxamide 30N

N-{3-[2,6-dimethylmorpholin-4-yl]-2-hydroxypropyl}-2,4-dimethyl-5-{[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]methyl}-1H-pyrrole-3-carboxamide 34N

5-[(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[2-hydroxy-3-(3-methyl-2,5-dioxoimidazolidin-1-yl)propyl]-2,4-dimethyl- 1H-pyrrole-3-carboxamide35N

N-[2-hydroxy-3-(3-methyl-2,5- dioxoimidazolidin-1-yl)propyl]-2,4-dimethyl-5-{(Z)-[2-oxo-5- (trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]methyl}-1H-pyrrole-3-carboxamide 36N

5-[(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[2-hydroxy-3-(3-methyl-2,5-dioxoimidazolidin-1-yl)propyl]-2,4-dimethyl- 1H-pyrrole-3-carboxamide37N

N-[3-(1,1-dioxidothiomorpholin-4-yl)-2-hydroxypropyl]-2,4-dimethyl-5-[(2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-1H- pyrrole-3-carboxamide 38N

N-[3-(1,1-dioxidothiomorpholin-4-yl)-2-hydroxypropyl]-5-[(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide 39N

5-[(5-chloro-2-oxo-1,2-dihydro-3H-indol-3- ylidene)methyl]-N-[3-(1,1-dioxidothiomorpholin-4-yl)-2-hydroxypropyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide 40N

5-[(5-bromo-2-oxo-1,2-dihydro-3H-indol-3- ylidene)methyl]-N-[3-(1,1-dioxidothiomorpholin-4-yl)-2-hydroxypropyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide 47N

5-(5-fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole- 3-carboxylic acid [2-hydroxy-3-([1,2,3]triazolo[4,5-b]pyridin-3-yloxy)- propyl]-amide 48N

5-(5-chloro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole- 3-carboxylic acid [2-hydroxy-3-([1,2,3]triazolo[4,5-b]pyridin-3-yloxy)- propyl]-amide 49N

2,4-dimethyl-5-(2-oxo-5- trifluoromethoxy-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-3-carboxylic acid[2-hydroxy-3-([1,2,3]triazolo[4,5- b]pyridin-3-yloxy)-propyl]-amide 50N

5-(5-fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole- 3-carboxylic acid[2-hydroxy-3-(3-oxy- benzotriazol-1-yl)-propyl]-amide 51N

5-(5-chloro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole- 3-carboxylic acid[2-hydroxy-3-(3-oxy- benzotriazol-1-yl)-propyl]-amide 52N

2,4-dimethyl-5-(2-oxo-5- trifluoromethoxy-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-3-carboxylic acid[2-hydroxy-3-(3-oxy-benzotriazol-1- yl)-propyl]-amide

Other representative compounds of the present invention are shown inTable 1c below.

TABLE 1c Cpd No. Structure Name 45N

5-(5-fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3- carboxylic acid(2-hydroxy-3-morpholin-4-yl- propyl)-methyl-amide; 45S

5-((Z)-5-fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3- carboxylic acid((S)-2-hydroxy-3-morpholin-4-yl- propyl)-methyl-amide 46S

5-((Z)-5-fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3- carboxylic acid((R)-2-hydroxy-3-morpholin-4-yl- propyl)-methyl-amide.

The compounds presented in Tables 1a-1c are exemplary only and are notto be construed as limiting the scope of this invention in any manner.

PREFERRED EMBODIMENTS

While the broadest definition is set forth in the Summary of theInvention, certain compounds of Formula (I) set forth below arepreferred.

-   A preferred group of compounds of Formula (I) is that wherein:-   R⁶ is selected from the group consisting of hydrogen and alkyl,    preferably hydrogen, methyl, ethyl, isopropyl, tert-butyl, isobutyl,    or n-butyl, more preferably hydrogen or methyl; and-   R⁷ is selected from the group consisting of hydrogen, alkyl, aryl,    heteroaryl, and —C(O)R¹⁷ wherein R¹⁷ is hydroxy, alkyl, cycloalkyl,    aryl, or heteroaryl, and more preferably R⁷ is hydrogen, methyl,    ethyl, isopropyl, n-, iso or tert-butyl, phenyl, benzoyl, acetyl or    carboxy, even more preferably methyl, hydrogen or phenyl.-   2. Another preferred group of compounds of Formula (I) is that    wherein:-   R⁶ is selected from the group consisting of hydrogen and alkyl,    preferably hydrogen, methyl, ethyl, isopropyl, tert-butyl, isobutyl,    or n-butyl, more preferably hydrogen or methyl, most preferably    methyl;-   R⁷ is selected from the group consisting of hydrogen, alkyl, aryl,    heteroaryl, and —C(O)R¹⁷ wherein R¹⁷ is hydroxy, alkyl or aryl, and    R⁷ is more preferably hydrogen, methyl, ethyl, isopropyl, n-, iso or    tert-butyl, phenyl, benzoyl, acetyl or carboxy, even more preferably    methyl, hydrogen or phenyl; and-   R³, R⁴, and R⁵ are hydrogen; and-   Z is aryl.-   3. Another preferred group of compounds of Formula (I) is that    wherein:-   R⁶ is selected from the group consisting of hydrogen and alkyl,    preferably hydrogen, methyl, ethyl, isopropyl, tert-butyl, isobutyl,    or n-butyl, more preferably hydrogen or methyl, most preferably    methyl;-   R⁷ is selected from the group consisting of hydrogen, alkyl, aryl,    heteroaryl, and —C(O)R¹⁷ wherein R¹⁷ is hydroxy, alkyl or aryl, and    R⁷ is more preferably hydrogen, methyl, ethyl, isopropyl, n-, iso or    tert-butyl, phenyl, benzoyl, acetyl or carboxy, even more preferably    methyl, hydrogen or phenyl, most preferably methyl; and-   R³, R⁴, and R⁵ are hydrogen; and-   Z is heteroaryl, preferably triazinyl, tetrazolyl, imidazolyl,    pyridinyl, pyrimidinyl or pyrazinyl.-   4. Another preferred group of compounds of Formula (I) is that    wherein:-   R⁶ is selected from the group consisting of hydrogen and alkyl,    preferably hydrogen, methyl, ethyl, isopropyl, tert-butyl, isobutyl,    or n-butyl, more preferably hydrogen or methyl, most preferably    methyl;-   R⁷ is selected from the group consisting of hydrogen, alkyl, aryl,    heteroaryl, and —C(O)R¹⁷ wherein R¹⁷ is hydroxy, alkyl or aryl, and    R⁷ is more preferably hydrogen, methyl, ethyl, isopropyl, n-, iso or    tert-butyl, phenyl, benzoyl, acetyl or carboxy, even more preferably    methyl, hydrogen or phenyl; and-   R³, R⁴, and R⁵ are hydrogen; and-   Z is heterocycle.-   5. Another preferred group of compounds of Formula (I) is that    wherein:-   R⁶ is selected from the group consisting of hydrogen and alkyl,    preferably hydrogen, methyl, ethyl, isopropyl, tert-butyl, isobutyl,    or n-butyl, more preferably hydrogen or methyl, most preferably    methyl;-   R⁷ is selected from the group consisting of hydrogen, alkyl, aryl,    heteroaryl, and —C(O)R¹⁷ wherein R¹⁷ is hydroxy, alkyl or aryl, and    R⁷ is more preferably hydrogen, methyl, ethyl, isopropyl, n−, iso or    tert-butyl, phenyl, benzoyl, acetyl or carboxy, even more preferably    methyl, hydrogen or phenyl, most preferably methyl; and-   R³, R⁴, and R⁵ are hydrogen; and-   Z is —NR¹⁵R¹⁶ wherein R¹⁵ and R¹⁶ combine to form heterocyclamino,    preferably piperidin-1-yl, N-methylpiperidin-1-yl, piperazin-1-yl,    N-methylpyrrolidin-1-yl, pyrrolidin-1-yl, morpholin-4-yl,    thiomorpholin-4-yl, thiomorpholino-1-oxide,    thiomorpholino-1,1-dioxide, 4ethyloxycarbonylmethylpiperazin-1-yl,    3-oxopiperazin-1-yl, imidazolidin-1-yl-2-one, pyrrolidin-1-yl-2-one,    2-oxohomopiperazin-1-yl, or tetrahydropyrimidin-1-yl-2-one, more    preferably morpholin-4-yl.-   5. Another preferred group of compounds of Formula (I) is that    wherein:-   R⁶ is selected from the group consisting of hydrogen and alkyl,    preferably hydrogen, methyl, ethyl, isopropyl, tert-butyl, isobutyl,    or n-butyl, more preferably hydrogen or methyl;-   R⁷ is selected from the group consisting of hydrogen, alkyl, aryl,    heteroaryl, and —C(O)R¹⁷ wherein R¹⁷ is hydroxy, alkyl or aryl, and    R⁷ is more preferably hydrogen, methyl, ethyl, isopropyl, n-, iso or    tert-butyl, phenyl, benzoyl, acetyl or carboxy, even more preferably    methyl, hydrogen or phenyl; and-   R³, R⁴, and R⁵ are hydrogen; and-   Z is —NR¹⁵R¹⁶ wherein R¹⁵ and R¹⁶ are alkyl, preferably    diethylamino, dimethylamino, or ethylamino.-   7. Within the above preferred and more preferred groups (1)-(6), an    even more preferred group of compounds is that wherein:    -   R¹ is hydrogen, alkyl, —C(O)NR¹²R¹³, unsubstituted cycloalkyl,        preferably hydrogen, 3,4-dimethoxyphenylaminocarbonyl,        4-methoxy-3-chlorophenyl-aminocarbonyl, even more preferably        hydrogen or methyl, most preferably hydrogen; and-   R² is hydrogen, cyano, halo, lower alkoxy, or —S(O)₂NR⁹R¹⁰ wherein    R⁹ is hydrogen and R¹⁰ is hydrogen, aryl or alkyl and is at the    5-position of the oxindole ring, preferably R² is hydrogen, chloro,    bromo, fluoro, methoxy, ethoxy, phenyl, dimethylaminosulfonyl,    3-chlorophenyl-aminosulfonyl, carboxy, methoxy, aminosulfonyl,    methylaminosulfonyl, phenylaminosulfonyl,    pyridin-3-yl-aminosulfonyl, dimethylaminosulfonyl,    isopropylamino-sulfonyl, more preferably hydrogen, fluoro, or bromo.    Most preferably R² is fluoro and is located at the 5-position of the    indolinone ring.    In the above preferred, more preferred and even more preferred    compounds the stereochemistry at the carbon atom carrying the    hydroxy group in the —CONHCH(R³)*CR⁴(OH)CR⁵Z chain and indicated by    a * is either RS, R, or S, more preferably S.

UTILITY

The PKs whose catalytic activity is modulated by the compounds of thisinvention include protein tyrosine kinases of which there are two types,receptor tyrosine kinases (RTKs) and cellular tyrosine kinases (CTKs),and serine-threonine kinases (STKs). RTK mediated signal transduction isinitiated by extracellular interaction with a specific growth factor(ligand), followed by receptor dimerization, transient stimulation ofthe intrinsic protein tyrosine kinase activity and phosphorylation.Binding sites are thereby created for intracellular signal transductionmolecules and lead to the formation of complexes with a spectrum ofcytoplasmic signaling molecules that facilitate the appropriate cellularresponse (e.g., cell division, metabolic effects on the extracellularmicroenvironment, etc.). See, Schlessinger and Ullrich, 1992, Neuron9:303-391.

It has been shown that tyrosine phosphorylation sites on growth factorreceptors function as high-affinity binding sites for SH2 (src homology)domains of signaling molecules. Fantl et al., 1992, Cell 69:413-423,Songyang et al., 1994, Mol. Cell. Biol. 14:2777-2785), Songyang et al.,1993, Cell 72:767-778, and Koch et al., 1991, Science 252:668-678.Several intracellular substrate proteins that associate with RTKs havebeen identified. They may be divided into two principal groups: (1)substrates that have a catalytic domain, and (2) substrates which lacksuch domain but which serve as adapters and associate with catalyticallyactive molecules. Songyang et al., 1993, Cell 72:767-778. Thespecificity of the interactions between receptors and SH2 domains oftheir substrates is determined by the amino acid residues immediatelysurrounding the phosphorylated tyrosine residue. Differences in thebinding affinities between SH2 domains and the amino acid sequencessurrounding the phosphotyrosine residues on particular receptors areconsistent with the observed differences in their substratephosphorylation profiles. Songyang et al., 1993, Cell 72:767-778. Theseobservations suggest that the function of each RTK is determined notonly by its pattern of expression and ligand availability but also bythe array of downstream signal transduction pathways that are activatedby a particular receptor. Thus, phosphorylation provides an importantregulatory step which determines the selectivity of signaling pathwaysrecruited by specific growth factor receptors, as well asdifferentiation factor receptors.

STKs, being primarily cytosolic, affect the internal biochemistry of thecell, often as a down-line response to a PTK event. STKs have beenimplicated in the signaling process which initiates DNA synthesis andsubsequent mitosis leading to cell proliferation.

Thus, PK signal transduction results in, among other responses, cellproliferation, differentiation, growth and metabolism. Abnormal cellproliferation may result in a wide array of disorders and diseases,including the development of neoplasia such as carcinoma, sarcoma,glioblastoma and hemangioma, disorders such as leukemia, psoriasis,arteriosclerosis, arthritis and diabetic retinopathy and other disordersrelated to uncontrolled angiogenesis and/or vasculogenesis.

A precise understanding of the mechanism by which the compounds of thisinvention inhibit PKs is not required in order to practice the presentinvention. However, while not hereby being bound to any particularmechanism or theory, it is believed that the compounds interact with theamino acids in the catalytic region of PKs. PKs typically possess abi-lobate structure wherein ATP appears to bind in the cleft between thetwo lobes in a region where the amino acids are conserved among PKs.Inhibitors of PKs are believed to bind by non-covalent interactions suchas hydrogen bonding, van der Waals forces and ionic interactions in thesame general region where the aforesaid ATP binds to the PKs. Morespecifically, it is thought that the 2-indolinone component of thecompounds of this invention binds in the general space normally occupiedby the adenine ring of ATP. Specificity of a particular molecule for aparticular PK may then arise as the result of additional interactionsbetween the various substituents on the 2-indolinone core and the aminoacid domains specific to particular PKs. Thus, different indolinonesubstituents may contribute to preferential binding to particular PKs.The ability to select compounds active at different ATP (or othernucleotide) binding sites makes the compounds of this invention usefulfor targeting any protein with such a site. The compounds disclosedherein thus have utility in in vitro assays for such proteins as well asexhibiting in vivo therapeutic effects through interaction with suchproteins.

Additionally, the compounds of the present invention provide atherapeutic approach to the treatment of many kinds of solid tumors,including but not limited to carcinomas, sarcomas including Kaposi'ssarcoma, erythroblastoma, glioblastoma, meningioma, astrocytoma,melanoma and myoblastoma. Treatment or prevention of non-solid tumorcancers such as leukemia are also contemplated by this invention.Indications may include, but are not limited to brain cancers, bladdercancers, ovarian cancers, gastric cancers, pancreas cancers, coloncancers, blood cancers, lung cancers and bone cancers.

Further examples, without limitation, of the types of disorders relatedto inappropriate PK activity that the compounds described herein may beuseful in preventing, treating and studying, are cell proliferativedisorders, fibrotic disorders and metabolic disorders.

Cell proliferative disorders, which may be prevented, treated or furtherstudied by the present invention include cancer, blood vesselproliferative disorders and mesangial cell proliferative disorders.

Blood vessel proliferative disorders refer to disorders related toabnormal vasculogenesis (blood vessel formation) and angiogenesis(spreading of blood vessels). While vasculogenesis and angiogenesis playimportant roles in a variety of normal physiological processes such asembryonic development, corpus luteum formation, wound healing and organregeneration, they also play a pivotal role in cancer development wherethey result in the formation of new capillaries needed to keep a tumoralive. Other examples of blood vessel proliferation disorders includearthritis, where new capillary blood vessels invade the joint anddestroy cartilage, and ocular diseases, like diabetic retinopathy, wherenew capillaries in the retina invade the vitreous, bleed and causeblindness.

Two structurally related RTKs have been identified to bind VEGF withhigh affinity: the fms-like tyrosine 1 (fit-I) receptor (Shibuya et al.,1990, Oncogene, 5:519-524; De Vries et al., 1992, Science, 255:989-991)and the KDR/FLK-1 receptor, also known as VEGF-R2. Vascular endothelialgrowth factor (VEGF) has been reported to be an endothelial cellspecific mitogen with in vitro endothelial cell growth promotingactivity. Ferrara & Henzel, 1989, Biochein. Biophys. Res. Comm.,161:851-858; Vaisman et al., 1990, J. Biol. Chem., 265:19461-19566.Information set forth in U.S. application Ser. Nos. 08/193,829,08/038,596 and 07/975,750, strongly suggest that VEGF is not onlyresponsible for endothelial cell proliferation, but also is the primeregulator of normal and pathological angiogenesis. See generally,Klagsburn & Soker, 1993, Current Biology, 3(10)699-702; Houck, et al.,1992, J. Biol. Chem., 267:26031-26037.

Normal vasculogenesis and angiogenesis play important roles in a varietyof physiological processes such as embryonic development, wound healing,organ regeneration and female reproductive processes such as follicledevelopment in the corpus luteum during ovulation and placental growthafter pregnancy. Folkman & Shing, 1992, J. Biological Chem.,267(16):10931-34. Uncontrolled vasculogenesis and/or angiogenesis hasbeen associated with diseases such as diabetes as well as with malignantsolid tumors that rely on vascularization for growth. Klagsburn & Soker,1993, Current Biology, 3(10):699-702; Folkham, 1991, J. Natl. CancerInst., 82:4-6; Weidner, et al., 1991, New Engl. J. Med., 324:1-5.

The surmised role of VEGF in endothelial cell proliferation andmigration during angiogenesis and vasculogenesis indicates an importantrole for the KDR/FLK-1 receptor in these processes. Diseases such asdiabetes mellitus (Folkman, 198, in XI ^(th) Congress of Thrombosis andHaemostasis (Verstraeta, et al., eds.), pp. 583-596, Leuven UniversityPress, Leuven) and arthritis, as well as malignant tumor growth mayresult from uncontrolled angiogenesis. See e.g., Folkman, 1971, N. Engl.J. Med., 285:1182-1186. The receptors to which VEGF specifically bindsare an important and powerful therapeutic target for the regulation andmodulation of vasculogenesis and/or angiogenesis and a variety of severediseases which involve abnormal cellular growth caused by suchprocesses. Plowman, et al., 1994, DN&P, 7(6):334-339. More particularly,the KDR/FLK-1 receptor's highly specific role in neovascularization makeit a choice target for therapeutic approaches to the treatment of cancerand other diseases which involve the uncontrolled formation of bloodvessels.

Thus, the present invention provides compounds capable of regulatingand/or modulating tyrosine kinase signal transduction includingKDR/FLK-1 receptor signal transduction in order to inhibit or promoteangiogenesis and/or vasculogenesis, that is, compounds that inhibit,prevent, or interfere with the signal transduced by KDR/FLK-1 whenactivated by ligands such as VEGF. Although it is believed that thecompounds of the present invention act on a receptor or other componentalong the tyrosine kinase signal transduction pathway, they may also actdirectly on the tumor cells that result from uncontrolled angiogenesis.

Although the nomenclature of the human and murine counterparts of thegeneric “flk-I” receptor differ, they are, in many respects,interchangeable. The murine receptor, Flk-1, and its human counterpart,KDR, share a sequence homology of 93.4% within the intracellular domain.Likewise, murine FLK-I binds human VEGF with the same affinity as mouseVEGF, and accordingly, is activated by the ligand derived from eitherspecies. Millauer et al., 1993, Cell, 72:835-846; Quinn et al., 1993,Proc. Natl. Acad. Sci. USA, 90:7533-7537. FLK-1 also associates with andsubsequently tyrosine phosphorylates human RTK substrates (e.g., PLC-γor p85) when co-expressed in 293 cells (human embryonal kidneyfibroblasts).

Models which rely upon the FLK-1 receptor therefore are directlyapplicable to understanding the KDR receptor. For example, use of themurine FLK-1 receptor in methods which identify compounds that regulatethe murine signal transduction pathway are directly applicable to theidentification of compounds which may be used to regulate the humansignal transduction pathway, that is, which regulate activity related tothe KDR receptor. Thus, chemical compounds identified as inhibitors ofKDR/FLK-1 in vitro, can be confirmed in suitable in vivo models. Both invivo mouse and rat animal models have been demonstrated to be ofexcellent value for the examination of the clinical potential of agentsacting on the KDR/FLK-1 induced signal transduction pathway.

Thus, the present invention provides compounds that regulate, modulateand/or inhibit vasculogenesis and/or angiogenesis by affecting theenzymatic activity of the KDR/FLK-1 receptor and interfering with thesignal transduced by KDR/FLK-1. Thus the present invention provides atherapeutic approach to the treatment of many kinds of solid tumorsincluding, but not limited to, glioblastoma, melanoma and Kaposi'ssarcoma, and ovarian, lung, mammary, prostate, pancreatic, colon andepidermoid carcinoma. In addition, data suggests the administration ofcompounds which inhibit the KDR/Flk-1 mediated signal transductionpathway may also be used in the treatment of hemangioma, restenois anddiabetic retinopathy.

Furthermore, this invention relates to the inhibition of vasculogenesisand angiogenesis by other receptor-mediated pathways, including thepathway comprising the fit-I receptor.

Receptor tyrosine kinase mediated signal transduction is initiated byextracellular interaction with a specific growth factor (ligand),followed by receptor dimerization, transient stimulation of theintrinsic protein tyrosine kinase activity and autophosphorylation.Binding sites are thereby created for intracellular signal transductionmolecules which leads to the formation of complexes with a spectrum ofcytoplasmic signalling molecules that facilitate the appropriatecellular response, e.g., cell division and metabolic effects to theextracellular microenvironment. See, Schlessinger and Ullrich, 1992,Neuron, 9:1-20.

The close homology of the intracellular regions of KDR/FLK-1 with thatof the PDGF-β receptor (50.3% homology) and/or the related fit-Ireceptor indicates the induction of overlapping signal transductionpathways. For example, for the PDGF-β receptor, members of the srcfamily (Twamley et al., 1993, Proc. Natl. Acad. Sci. USA, 90:7696-7700),phosphatidylinositol-3′-kinase (Hu et al., 1992, Mol. Cell. Biol.,12:981-990), phospholipase cγ (Kashishian & Cooper, 1993, Mol. Cell.Biol., 4:49-51), ras-GTPase-activating protein, (Kashishian et al.,1992, EMBO J., 11:1373-1382), PTP-ID/syp (Kazlauskas et al., 1993, Proc.Natl. Acad. Sci. USA, 10 90:6939-6943), Grb2 (Arvidsson et al., 1994,Mol. Cell. Biol., 14:6715-6726), and the adapter molecules Shc and Nck(Nishimura et al., 1993, Mol. Cell. Biol., 13:6889-6896), have beenshown to bind to regions involving different autophosphorylation sites.See generally, Claesson-Welsh, 1994, Prog. Growth Factor Res., 5:37-54.Thus, it is likely that signal transduction pathways activated byKDR/FLK-1 include the ras pathway (Rozakis et al., 1992, Nature,360:689-692), the PI-3′-kinase, the src-mediated and the plcγ-mediatedpathways. Each of these pathways may play a critical role in theangiogenic and/or vasculogenic effect of KDR/FLK-1 in endothelial cells.Consequently, a still further aspect of this invention relates to theuse of the organic compounds described herein to modulate angiogenesisand vasculogenesis as such processes are controlled by these pathways.

Conversely, disorders related to the shrinkage, contraction or closingof blood vessels, such as restenosis, are also implicated and may betreated or prevented by the methods of this invention.

Fibrotic disorders refer to the abnormal formation of extracellularmatrices. Examples of fibrotic disorders include hepatic cirrhosis andmesangial cell proliferative disorders. Hepatic cirrhosis ischaracterized by the increase in extracellular matrix constituentsresulting in the formation of a hepatic scar. An increased extracellularmatrix resulting in a hepatic scar can also be caused by a viralinfection such as hepatitis. Lipocytes appear to play a major role inhepatic cirrhosis. Other fibrotic disorders implicated includeatherosclerosis.

Mesangial cell proliferative disorders refer to disorders brought aboutby abnormal proliferation of mesangial cells. Mesangial proliferativedisorders include various human renal diseases such asglomerulonephritis, diabetic nephropathy and malignant nephrosclerosisas well as such disorders as thrombotic microangiopathy syndromes,transplant rejection, and glomerulopathies. The RTK PDGFR has beenimplicated in the maintenance of mesangial cell proliferation. Floege etal., 1993, Kidney International 43:47 S-54S.

Many cancers are cell proliferative disorders and, as noted previously,PKs have been associated with cell proliferative disorders. Thus, it isnot surprising that PKs such as, for example, members of the RTK familyhave been associated with the development of cancer. Some of thesereceptors, like EGFR (Tuzi et al., 1991, Br. J. Cancer 63:227-233, Torpet al., 1992, APMIS 100:713-719) HER2/neu (Slamon et al., 1989, Science244:707-712) and PDGF-R (Kumabe et al., 1992, Oncogene, 7:627-633) areover-expressed in many tumors and/or persistently activated by autocrineloops. In fact, in the most common and severe cancers these receptorover-expressions (Akbasak and Suner-Akbasak et al., 1992, J. Neurol.Sci., 111:119-133, Dickson et al., 1992, Cancer Treatment Res.61:249-273, Korc et al., 1992, J. Clin. Invest. 90:1352-1360) andautocrine loops (Lee and Donoghue, 1992, J. Cell. Biol., 118:1057-1070,Korc et al., supra, Akbasak and Suner-Akbasak et al., supra) have beendemonstrated. For example, EGFR has been associated with squamous cellcarcinoma, astrocytoma, glioblastoma, head and neck cancer, lung cancerand bladder cancer. HER2 has been associated with breast, ovarian,gastric, lung, pancreas and bladder cancer. PDGFR has been associatedwith glioblastoma and melanoma as well as lung, ovarian and prostatecancer. The RTK c-met has also been associated with malignant tumorformation. For example, c-met has been associated with, among othercancers, colorectal, thyroid, pancreatic, gastric and hepatocellularcarcinomas and lymphomas. Additionally c-met has been linked toleukemia. Over-expression of the c-met gene has also been detected inpatients with Hodgkins disease and Burkitts disease.

IGF-IR, in addition to being implicated in nutritional support and intype-II diabetes, has also been associated with several types ofcancers. For example, IGF-I has been implicated as an autocrine growthstimulator for several tumor types, e.g. human breast cancer carcinomacells (Arteaga et al., 1989, J. Clin. Invest. 84:1418-1423) and smalllung tumor cells (Macauley et al., 1990, Cancer Res., 50:2511-2517). Inaddition, IGF-I, while integrally involved in the normal growth anddifferentiation of the nervous system, also appears to be an autocrinestimulator of human gliomas. Sandberg-Nordqvist et al., 1993, CancerRes. 53:2475-2478. The importance of IGF-IR and its ligands in cellproliferation is further supported by the fact that many cell types inculture (fibroblasts, epithelial cells, smooth muscle cells,T-lymphocytes, myeloid cells, chondrocytes and osteoblasts (the stemcells of the bone marrow)) are stimulated to grow by IGF-I. Goldring andGoldring, 1991, Eukaryotic Gene Expression, 1:301-326. Baserga andCoppola suggest that IGF-IR plays a central role in the mechanism oftransformation and, as such, could be a preferred target for therapeuticinterventions for a broad spectrum of human malignancies. Baserga, 1995,Cancer Res., 55:249-252, Baserga, 1994, Cell 79:927-930, Coppola et al.,1994, Mol. Cell. Biol., 14:4588-4595.

STKs have been implicated in many types of cancer including, notably,breast cancer (Cance, et al., Int. J. Cancer, 54:571-77 (1993)).

The association between abnormal PK activity and disease is notrestricted to cancer. For example, RTKs have been associated withdiseases such as psoriasis, diabetes mellitus, endometriosis,angiogenesis, atheromatous plaque development, Alzheimer's disease,restenosis, von Hippel-Lindau disease, epidermal hyperproliferation,neurodegenerative diseases, age-related macular degeneration andhemangiomas. For example, EGFR has been indicated in corneal and dermalwound healing. Defects in Insulin-R and IGF-1R are indicated in type-IIdiabetes mellitus. A more complete correlation between specific RTKs andtheir therapeutic indications is set forth in Plowman et al., 1994, DN&P7:334-339.

As noted previously, not only RTKs but CTKs including, but not limitedto, src, abl, fps, yes, fyn, lyn, Ick, blk, hck, fgr and yrk (reviewedby Bolen et al., 1992, FASEB J., 6:3403-3409) are involved in theproliferative and metabolic signal transduction pathway and thus couldbe expected, and have been shown, to be involved in many PTK-mediateddisorders to which the present invention is directed. For example,mutated src (v-src) has been shown to be an oncoprotein (pp60^(v-src))in chicken. Moreover, its cellular homolog, the proto-oncogenepp60^(c-src) transmits oncogenic signals of many receptors.Over-expression of EGFR or HER2/neu in tumors leads to the constitutiveactivation of pp60^(c-src), which is characteristic of malignant cellsbut absent in normal cells. On the other hand, mice deficient in theexpression of c-src exhibit an osteopetrotic phenotype, indicating a keyparticipation of c-src in osteoclast function and a possible involvementin related disorders.

Similarly, Zap70 has been implicated in T-cell signaling which mayrelate to autoimmune disorders.

STKs have been associated with inflammation, autoimmune disease,immunoresponses, and hyperproliferation disorders such as restenosis,fibrosis, psoriasis, osteoarthritis and rheumatoid arthritis.

PKs have also been implicated in embryo implantation. Thus, thecompounds of this invention may provide an effective method ofpreventing such embryo implantation and thereby be useful as birthcontrol agents. Additional disorders which may be treated or preventedusing the compounds of this invention are immunological disorders suchas autoimmune disease, AIDS and cardiovasular disorders such asatherosclerosis.

Finally, both RTKs and CTKs are currently suspected as being involved inhyperimmune disorders.

The compounds and data presented are not to be construed as limiting thescope of this invention in any manner whatsoever.

Administration and Pharmaceutical Composition

A compound of the present invention or a pharmaceutically acceptablesalt thereof, can be administered as such to a human patient or can beadministered in pharmaceutical compositions in which the foregoingmaterials are mixed with suitable carriers or excipient(s). Techniquesfor formulation and administration of drugs may be found in “Remington'sPharmacological Sciences,” Mack Publishing Co., Easton, Pa., latestedition.

As used herein, “administer” or “administration” refers to the deliveryof a compound of Formula (I) or a pharmaceutically acceptable saltthereof or of a pharmaceutical composition containing a compound ofFormula (I) or a pharmaceutically acceptable salt thereof of thisinvention to an organism for the purpose of prevention or treatment of aPK-related disorder.

Suitable routes of administration may include, without limitation, oral,rectal, transmucosal or intestinal administration or intramuscular,subcutaneous, intramedullary, intrathecal, direct intraventricular,intravenous, intravitreal, intraperitoneal, intranasal, or intraocularinjections. The preferred routes of administration are oral andparenteral.

Alternatively, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto a solid tumor, often in a depot or sustained release formulation.

Furthermore, one may administer the drug in a targeted drug deliverysystem, for example, in a liposome coated with tumor-specific antibody.The liposomes will be targeted to and taken up selectively by the tumor.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the compounds of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks' solution, Ringer's solution, or physiological saline buffer.For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be formulated by combiningthe active compounds with pharmaceutically acceptable carriers wellknown in the art. Such carriers enable the compounds of the invention tobe formulated as tablets, pills, lozenges, dragees, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient. Pharmaceutical preparations for oral use can be made using asolid excipient, optionally grinding the resulting mixture, andprocessing the mixture of granules, after adding other suitableauxiliaries if desired, to obtain tablets or dragee cores. Usefulexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol, cellulose preparations such as,for example, maize starch, wheat starch, rice starch and potato starchand other materials such as gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinyl-pyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginicacid. A salt such as sodium alginate may also be used.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with a fillersuch as lactose, a binder such as starch, and/or a lubricant such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. Stabilizers may be added in these formulations, also.

Pharmaceutical compositions which may also be used include hard gelatincapsules. As a non-limiting example, the active compound capsule oraldrug product formulation may be as 50 and 200 mg dose strengths. The twodose strengths are made from the same granules by filling into differentsize hard gelatin capsules, size 3 for the 50 mg capsule and size 0 forthe 200 mg capsule. The composition of the formulation may be, forexample, as indicated in Table 2.

TABLE 2 Concentration Amount in Amount in Ingredient in Granulation 50mg 200 mg Name/Grade (% w/w) Capsule (mg) Capsule (mg) Active CompoundNF 65.0 50.0 200.0 Mannitol NF 23.5 18.1 72.4 Croscarmellose 6.0 4.618.4 sodium NF Povidone K 30 NF 5.0 3.8 15.2 Magnesium 0.5 0.38 1.52stearate NF Capsule, Swedish Size 3 Size 0 yellow NF

The capsules may be packaged into brown glass or plastic bottles toprotect the active compound from light. The containers containing theactive compound capsule formulation must be stored at controlled roomtemperature (15-30° C.).

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray using a pressurized pack or a nebulizer and a suitable propellant,e.g., without limitation, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetra-fluoroethane or carbon dioxide. Inthe case of a pressurized aerosol, the dosage unit may be controlled byproviding a valve to deliver a metered amount. Capsules and cartridgesof, for example, gelatin for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

The compounds may also be formulated for parenteral administration,e.g., by bolus injection or continuous infusion. Formulations forinjection may be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with an added preservative. The compositions maytake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulating materials such assuspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of a water soluble form, such as, without limitation,a salt, of the active compound. Additionally, suspensions of the activecompounds may be prepared in a lipophilic vehicle. Suitable lipophilicvehicles include fatty oils such as sesame oil, synthetic fatty acidesters such as ethyl oleate and triglycerides, or materials such asliposomes. Aqueous injection suspensions may contain substances whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers and/or agents that increase the solubilityof the compounds to allow for the preparation of highly concentratedsolutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, using, e.g., conventional suppositorybases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as depot preparations. Such long acting formulationsmay be administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. A compound of thisinvention may be formulated for this route of administration withsuitable polymeric or hydrophobic materials (for instance, in anemulsion with a pharmacologically acceptable oil), with ion exchangeresins, or as a sparingly soluble derivative such as, withoutlimitation, a sparingly soluble salt.

A non-limiting example of a pharmaceutical carrier for the hydrophobiccompounds of the invention is a cosolvent system comprising benzylalcohol, a nonpolar surfactant, a water-miscible organic polymer and anaqueous phase such as the VPD co-solvent system. VPD is a solution of 3%w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80,and 65% w/v polyethylene glycol 300, made up to volume in absoluteethanol. The VPD co-solvent system (VPD:D5W) consists of VPD diluted 1:1with a 5% dextrose in water solution. This co-solvent system dissolveshydrophobic compounds well, and itself produces low toxicity uponsystemic administration. Naturally, the proportions of such a co-solventsystem may be varied considerably without destroying its solubility andtoxicity characteristics. Furthermore, the identity of the co-solventcomponents may be varied: for example, other low-toxicity nonpolarsurfactants may be used instead of Polysorbate 80, the fraction size ofpolyethylene glycol may be varied, other biocompatible polymers mayreplace polyethylene glycol, e.g., polyvinyl pyrrolidone, and othersugars or polysaccharides may substitute for dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceuticalcompounds may be employed. Liposomes and emulsions are well knownexamples of delivery vehicles or carriers for hydrophobic drugs. Inaddition, certain organic solvents such as dimethylsulfoxide also may beemployed, although often at the cost of greater toxicity.

Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid hydrophobic polymerscontaining the therapeutic agent. Various sustained-release materialshave been established and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the therapeuticreagent, additional strategies for protein stabilization may beemployed.

The pharmaceutical compositions herein also may comprise suitable solidor gel phase carriers or excipients. Examples of such carriers orexcipients include, but are not limited to, calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols.

Many of the PK modulating compounds of the invention may be provided asphysiologically acceptable salts wherein the claimed compound may formthe negatively or the positively charged species. Examples of salts inwhich the compound forms the positively charged moiety include, withoutlimitation, quaternary ammonium (defined elsewhere herein), salts suchas the hydrochloride, sulfate, carbonate, lactate, tartrate, malate,maleate, succinate wherein the nitrogen atom of the quaternary ammoniumgroup is a nitrogen of the selected compound of this invention which hasreacted with the appropriate acid. Salts in which a compound of thisinvention forms the negatively charged species include, withoutlimitation, the sodium, potassium, calcium and magnesium salts formed bythe reaction of a carboxylic acid group in the compound with anappropriate base (e.g. sodium hydroxide (NaOH), potassium hydroxide(KOH), Calcium hydroxide (Ca(OH)₂), etc.).

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in anamount sufficient to achieve the intended purpose, e.g., the modulationof PK activity or the treatment or prevention of a PK-related disorder.

More specifically, a therapeutically effective amount means an amount ofcompound effective to prevent, alleviate or ameliorate symptoms ofdisease or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any compound used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromcell culture assays. Then, the dosage can be formulated for use inanimal models so as to achieve a circulating concentration range thatincludes the IC₅₀ as determined in cell culture (i.e., the concentrationof the test compound which achieves a half-maximal inhibition of the PKactivity). Such information can then be used to more accuratelydetermine useful doses in humans.

Toxicity and therapeutic efficacy of the compounds described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., by determining the IC₅₀ and the LD₅₀ (bothof which are discussed elsewhere herein) for a subject compound. Thedata obtained from these cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage mayvary depending upon the dosage form employed and the route ofadministration utilized. The exact formulation, route of administrationand dosage can be chosen by the individual physician in view of thepatient's condition. (See e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active species which are sufficient to maintain thekinase modulating effects. These plasma levels are referred to asminimal effective concentrations (MECs). The MEC will vary for eachcompound but can be estimated from in vitro data, e.g., theconcentration necessary to achieve 50-90% inhibition of a kinase may beascertained using the assays described herein. Dosages necessary toachieve the MEC will depend on individual characteristics and route ofadministration. HPLC assays or bioassays can be used to determine plasmaconcentrations.

Dosage intervals can also be determined using MEC value. Compoundsshould be administered using a regimen that maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%.

At present, the therapeutically effective amounts of compounds ofFormulas I, Ia, or II may range from approximately 25 mg/m² to 1500mg/m² per day; preferably about 3 mg/m²/day. Even more preferably 50mg/qm qd till 400 mg/qd.

In cases of local administration or selective uptake, the effectivelocal concentration of the drug may not be related to plasmaconcentration and other procedures known in the art may be employed todetermine the correct dosage amount and interval.

The amount of a composition administered will, of course, be dependenton the subject being treated, the severity of the affliction, the mannerof administration, the judgment of the prescribing physician, etc.

The compositions may, if desired, be presented in a pack or dispenserdevice, such as an FDA approved kit, which may contain one or more unitdosage forms containing the active ingredient. The pack may for examplecomprise metal or plastic foil, such as a blister pack. The pack ordispenser device may be accompanied by instructions for administration.The pack or dispenser may also be accompanied by a notice associatedwith the container in a form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals, which noticeis reflective of approval by the agency of the form of the compositionsor of human or veterinary administration. Such notice, for example, maybe of the labeling approved by the U.S. Food and Drug Administration forprescription drugs or of an approved product insert. Compositionscomprising a compound of the invention formulated in a compatiblepharmaceutical carrier may also be prepared, placed in an appropriatecontainer, and labeled for treatment of an indicated condition. Suitableconditions indicated on the label may include treatment of a tumor,inhibition of angiogenesis, treatment of fibrosis, diabetes, and thelike.

The present invention can be administered with a CMC suspension vehicle.An exemplary CMC suspension is listed below in Table 3.

Component Concentration % (w/v) API * Carboxymethylcellulose sodium, USP0.5 (Medium grade) Sodium Chloride, USP/NF 0.9 Polysorbate 80, NF 0.4Benzyl Alcohol, NF 0.9 Water, deionized qs. 100 mL * Dependent onconcentration (date) required.

A protocol for a 1.0 Lit of CMC suspension vehicle is as follows.Calculate the appropriate amount of excipients required to make thevehicle formulation using the table showing the composition of vehicleformulation and the batch size. Weigh a suitable empty container, suchas a clean wide mouthed glass bottle, or a polyethylene bottle. Addabout 600 mL of water to the container. Weigh carboxymethylcellulosesodium (5 gms) and transfer to the container. Stir using a magnetic stirbar or a laboratory stirrer with propeller until homogenous (about 2-3hours). Weigh NaCl and add to the container. Continue mixing untildissolved (about 10 mins). Add polysorbate-80. Mix until the solution ishomogenous (about 20 mins). Add benzyl alcohol. Mix until the solutionis homogenous (about 10 mins). Add the remaining water to bring up theweight of the solution to the required batch size either by weight orvolume (1010 gms or 1000 mL, density at 22° C. is 1.01). Store at 2-8°C. (under refrigeration).

The suspension formulation can be manufactured as follows. Grind the APIusing a mortar and pestle to obtain a homogenous looking powder withsmall particulate size (no chunks or large particulates—ideally shouldpass through a US Standard Sieve >80 i.e. <180 μm size). Weigh thecalculated amount of API into the container. Add about 90% of the totalrequired amount of (CMC suspension vehicle) into the container. Suspendcompounds in the vehicle using a laboratory stirrer with propeller orequivalent. The diameter of the propeller blades should match thediameter of the bottom of the container to ensure efficient mixing. Stirat 50 rpm for 30 mins or until the drug is well suspended. Add thevehicle formulation to “qs” (bring up the water) (quality sufficient) tothe appropriate weight corresponding to the batch size. Stir at 50 rpmfor additional 30 mins. Aliquot the suspension immediately to ambercolored glass or polypropylene containers. Containers to be protectedfrom light. Stir at 2-8° C. (under refrigeration, do not freeze).

It is also an aspect of this invention that a compound described herein,or its salt or prodrug, might be combined with other chemotherapeuticagents for the treatment of the diseases and disorders discussed above.For instance, a compound, salt or prodrug of this invention might becombined with alkylating agents such as fluorouracil (5-FU) alone or infurther combination with leukovorin; or other alkylating agents such as,without limitation, other pyrimidine analogs such as UFT, capecitabine,gemcitabine and cytarabine, the alkyl sulfonates, e.g., busulfan (usedin the treatment of chronic granulocytic leukemia), improsulfan andpiposulfan; aziridines, e.g., benzodepa, carboquone, meturedepa anduredepa; ethyleneimines and methylmelamines, e.g., altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylolmelamine; and the nitrogenmustards, e.g., chlorambucil (used in the treatment of chroniclymphocytic leukemia, primary macroglobulinemia and non-Hodgkin'slymphoma), cyclophosphamide (used in the treatment of Hodgkin's disease,multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lungcancer, Wilm's tumor and rhabdomyosarcoma), estramustine, ifosfamide,novembrichin, prednimustine and uracil mustard (used in the treatment ofprimary thrombocytosis, non-Hodgkin's lymphoma, Hodgkin's disease andovarian cancer); and triazines, e.g., dacarbazine (used in the treatmentof soft tissue sarcoma).

A compound, salt or prodrug of this invention can also be used incombination with other antimetabolite chemotherapeutic agents such as,without limitation, folic acid analogs, e.g. methotrexate (used in thetreatment of acute lymphocytic leukemia, choriocarcinoma, mycosisfungiodes breast cancer, head and neck cancer and osteogenic sarcoma)and pteropterin; and the purine analogs such as mercaptopurine andthioguanine which find use in the treatment of acute granulocytic, acutelymphocytic and chronic granulocytic leukemias.

It is contemplated that a compound, salt or prodrug of this inventioncan also be used in combination with natural product basedchemotherapeutic agents such as, without limitation, the vincaalkaloids, e.g., vinblastin (used in the treatment of breast andtesticular cancer), vincristine and vindesine; the epipodophylotoxins,e.g., etoposide and teniposide, both of which are useful in thetreatment of testicular cancer and Kaposi's sarcoma; the antibioticchemotherapeutic agents, e.g., daunorubicin, doxorubicin, epirubicin,mitomycin (used to treat stomach, cervix, colon, breast, bladder andpancreatic cancer), dactinomycin, temozolomide, plicamycin, bleomycin(used in the treatment of skin, esophagus and genitourinary tractcancer); and the enzymatic chemotherapeutic agents such asL-asparaginase.

In addition to the above, a compound, salt or prodrug of this inventioncould also be used in combination with the platinum coordinationcomplexes (cisplatin, etc.); substituted ureas such as hydroxyurea;methylhydrazine derivatives, e.g., procarbazine; adrenocorticalsuppressants, e.g., mitotane, aminoglutethimide; and hormone and hormoneantagonists such as the adrenocorticosteriods (e.g., prednisone),progestins (e.g., hydroxyprogesterone caproate); estrogens (e.g.,diethylstilbesterol); antiestrogens such as tamoxifen; androgens, e.g.,testosterone propionate; and aromatase inhibitors such as anastrozole.

Finally, it is also contemplated that the combination of a compound ofthis invention will be effective in combination with mitoxantrone,paclitaxel, cyclooxygenase-2 inhibitors known in the art, in particularCelebrex®, Paracoxib®, Vioxx®, Abbott's Cox-189 disclosed in PCTPublication No. WO 99/11605, topoisomerase inhibitors such asCamptosar®, Her-2 receptor antagonist such as Herceptin®, endostatin,Gleevac®, ImClone VEGF receptor antagonist IMC C225® for the treatmentof solid tumor cancers or leukemias such as, without limitation, acutemyelogenous (non-lymphocytic) leukemia.

General Synthetic Procedure

The following general methodology may be employed to prepare thecompounds of this invention:

The appropriately substituted 2-oxindole (1 equiv.), the appropriatelysubstituted 3-carboxy-5-formylpyrrole (1.2 equiv.) and a base (0.1equiv.) are mixed in a solvent (1-2 ml/mmol 2-oxindole) and the mixtureis then heated for from about 2 to about 12 hours. After cooling, theprecipitate that forms is filtered, washed with cold ethanol or etherand vacuum dried to give corresponding5-(2-oxo-1,2-dihydroindol-(3Z)-ylidenemethyl)-1-H-pyrrole-3-carboxylicacid. If no precipitate forms, the reaction mixture is concentrated andthe residue is triturated with dichloromethane/ether, the resultingsolid is collected by filtration and then dried. The product mayoptionally be further purified by chromatography.

The base may be an organic or an inorganic base. If an organic base isused, preferably it is a nitrogen base. Examples of organic nitrogenbases include, but are not limited to, diisopropylamine, trimethylamine,triethylamine, aniline, pyridine, 1,8-diazabicyclo[5.4.1]undec-7-ene,pyrrolidine and piperidine.

Examples of inorganic bases are, without limitation, ammonia, alkalimetal or alkaline earth hydroxides, phosphates, carbonates,bicarbonates, bisulfates and amides. The alkali metals include, lithium,sodium and potassium while the alkaline earths include calcium,magnesium and barium.

In a presently preferred embodiment of this invention, when the solventis a protic solvent, such as water or alcohol, the base is an alkalimetal or an alkaline earth inorganic base, preferably, a alkali metal oran alkaline earth hydroxide.

It will be clear to those skilled in the art, based both on knowngeneral principles of organic synthesis and on the disclosures hereinwhich base would be most appropriate for the reaction contemplated.

The solvent in which the reaction is carried out may be a protic or anaprotic solvent, preferably it is a protic solvent. A “protic solvent”is a solvent which has hydrogen atom(s) covalently bonded to oxygen ornitrogen atoms which renders the hydrogen atoms appreciably acidic andthus capable of being “shared” with a solute through hydrogen bonding.Examples of protic solvents include, without limitation, water andalcohols.

An “aprotic solvent” may be polar or non-polar but, in either case, doesnot contain acidic hydrogens and therefore is not capable of hydrogenbonding with solutes. Examples, without limitation, of non-polar aproticsolvents, are pentane, hexane, benzene, toluene, methylene chloride andcarbon tetrachloride. Examples of polar aprotic solvents are chloroform,tetrahydro-furan, dimethylsulfoxide and dimethylformamide.

In a presently preferred embodiment of this invention, the solvent is aprotic solvent, preferably water or an alcohol such as ethanol.

The reaction is carried out at temperatures greater than roomtemperature. The temperature is generally from about 30° C. to about150° C., preferably about 80° C. to about 100° C., most preferable about60° C. to about 85° C., which is about the boiling point of ethanol. By“about” is meant that the temperature range is preferably within 10degrees Celcius of the indicated temperature, more preferably within 5degrees Celcius of the indicated temperature and, most preferably,within 2 degrees Celcius of the indicated temperature. Thus, forexample, by “about 75° C.” is meant 75° C.±10° C., preferably 75° C.±5°C. and most preferably, 75° C.±2° C.

2-Oxindoles and 3-carboxy-5-formylpyrrole, may be readily synthesizedusing techniques well known in the chemical arts using readily availablestarting materials.

Coupling of a5-(2-oxo-1,2-dihydroindol-(3Z)-ylidenemethyl)-1-H-pyrrole-3-carboxylicacid with an amine of formula ZCH(R⁵)—CR⁴(OH)—CHR³NH₂ in an organicsolvent such as dimethylformamide, tetrahydrofuran, and the like and inthe presence of a suitable coupling agent such asdicyclohexylcarbodiimide, DEAD, EDC and HOBt then provides a compound ofFormula (I). Amines of formula ZCH(R⁵)—CR⁴(OH)—CHR³NH₂ are commerciallyavailable or they can be prepared by method well known in the art. Somesuch procedures are described herein below.

It will be appreciated by those skilled in the art that other syntheticpathways for forming the compounds of the invention are available andthat the following is offered by way of example and not limitation.

EXAMPLES

The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and to practice thepresent invention. They should not be considered as limiting the scopeof the invention, but merely as being illustrative and representativethereof.

Synthetic Examples Example 1 Synthesis of5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidene-methyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid

Step 1

Dimethylformamide (25 mL, 3 eq.) was cooled with stirring in an icebath. To this was added POCl₃ (1.1 eq., 10.8 mL). After 30 minutes, asolution of the 3,5-dimethyl-4-ethylester pyrrole (17.7 g, 105.8 mmol)in DMF (2M, 40 mL) was added to the reaction and stirring continued.After 2 hour, the reaction was diluted with water (250 mL) and basifiedto pH=11 with 1 N aqueous NaOH. The white solid was removed byfiltration, rinsing with water and then hexanes and dried to afford5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid ethyl ester (19.75 g,95%) as a tan solid. ¹H NMR (360 MHz, DMSO-d6) δ 12.11 (br s, 1H, NH),9.59 (s, 1H, CHO), 4.17 (q, J=6.7 Hz, 2H, OCH ₂CH₃), 2.44 (s, 3H, CH ₃),2.40 (s, 3H, CH ₃), 1.26 (d, J=6.7 Hz, 3H, OCH₂CH ₃).

Step 2

5-Formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid ethyl ester (2 g, 10mmol) was added to a solution of potassium hydroxide (3 g, 53 mmol)dissolved in methanol (3 mL) and water (10 mL). The mixture was refluxedfor 3 hours, cooled to room temperature and acidified with 6 Nhydrochloric acid to pH 3. The solid was collected by filtration, washedwith water and dried in a vacuum oven overnight to give5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (1.6 g, 93%). ¹H NMR(300 MHz, DMSO-d6) δ 12.09 (s, br, 2H, NH & COOH), 9.59 (s, 1H, CHO),2.44 (s, 3H, CH₃), 2.40 (s, 3H, CH₃).

Step 3

5-Fluoroisatin (8.2 g, 49.7 mmol) was dissolved in 50 mL of hydrazinehydrate and refluxed for 1 hour. The reaction mixtures were then pouredin ice water. The precipitate was then filtered, washed with water anddried under vacuum oven to give 5-fluoro-2-oxindole (7.5 g).

Step 4

The reaction mixture of 5-fluorooxindole (100 mg, 0.66 mmol),5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (133 mg, 0.79 mmol),and 10 drops of piperidine in ethanol (3 mL) was stirred at 60° C.overnight and filtered. The solid washed with 1 M of aqueoushydrochloride solution, water, and dried to afford5-(5-fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (201 mg, quantitative) as a yellow solid. MS m/z (relativeintensity, %) 299 ([M−1]^(+.), 100).

Example 2 Synthesis of5-(5-Fluoro-2-oxo-1,2-dihydro-indol-3-ylidene-methyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (3-diethylamino-2-hydroxy-propyl)-amide

Step 1

To 2-chloromethyloxirane (95 g, 1.03 mole) was added a mixture of water(3.08 g, 0.17 mole) and diethylamine (106.2 mL, 1.03 mole) at 30° C. Thereaction mixture was then stirred at 28-35° C. for 6 hour and cooled to20-25° C. to give 1-chloro-3-diethylamino-propan-2-ol.

Step 2

A solution of sodium hydroxide (47.9 g, 1.2 mole) in 78 mL water wasadded 1-chloro-3-diethylamino-propan-2-ol. The resultant was stirred at20-25° C. for 1 hour, diluted with 178 mL of water and extracted withether twice. The combined ether solution was dried with solid potassiumhydroxide and evaporated to give 135 g of crude product which waspurified by fraction distillation to give pure glycidyldiethylamine (98g, 76%) as an oil.

Step 3

To the ice-cold solution of ammonium hydroxide (25 mL, 159 mmole) of 25%(w/w) was added glycidyldiethylamine dropwise (3.2 g, 24.8 mmol) over 10minutes. The reaction mixture was stirred at 0-5° C. for 1 hour and thenroom temperature for 14 hours. The resulting reaction mixture wasevaporated and distilled (84-90° C. at 500-600 mT) to yield1-amino-3-diethylamino-propan-2-ol (3.3 g, 92%). MS m/z 147([M+1]^(+.)).

Step 4

To the solution of 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(100 mg, 0.43 mmol), EDC (122.7 mg, 0.64 mmol) and HOBt (86.5 mg, 0.64mmol) in 1.0 mL of DMF was added 1-amino-3-diethylamino-propan-2-ol(93.2 mg, 0.64 mmol). The resulting reaction solution was stirred atroom temperature overnight and evaporated. The residue was suspended in10 mL of water and filtered. The solid washed with saturated sodiumbicarbonate and water and dried in a high vacuum oven overnight to givecrude product which was purified on column chromatography eluting with6% methanol-dichlormethane containing triethylamine (2 drops/100 mL of6% methanol-dichloromethane) to give5-(5-fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (3-diethylamino-2-hydroxy-propyl)-amide (62 mg, 34%) as a yellowsolid. ¹H NMR (400 MHz, DMSO-d6) δ 13.70 (s, 1H, NH-1′), 10.90 (s, 1H,NH-1), 7.76 (dd, J=2.38, 9.33 Hz, 1H, H-4), 7.72 (s, 1H, vinyl-H), 7.60(m, br., 1H, CONHCH₂CH(OH)—CH₂N(C₂H₅)₂-4′), 6.93 (dt, J=2.38, 8.99 Hz,1H, H-5), 6.85 (dd, J=4.55, 8.99 Hz, 1H, H-6), 3.83 (m, br, 1H, OH),3.33 (m, 4H), 2.67 (m, br, 5H), 2.46 (s, 3H, CH₃), 2.44 (s, 3H, CH₃),1.04 (m, br, 6H, CH₃×2). MS m/z (relative intensity, %) 427 ([M+1]^(+.),100).

Example 3 Synthesis of5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidene-methyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-hydroxy-3-morpholin-4-yl-propyl)-amide

Step 1

A mixture of morpholine (2.6 mL, 30 mmol) and epichlorohydrin (2.35 ml,30 mmol) in ethanol (50 mL) was stirred at 70° C. overnight. Afterremoving the solvent, the residue was diluted with methylene chloride(50 mL). The clear solid precipitated was collected by vacuum filtrationto give 1-chloro-3-morpholin-4-yl-propan-2-ol (2.0 g, 37%). ¹H NMR(DMSO-d₆) δ 3.49 (t, J=4.8 Hz, 2H), 3.60 (t, J=4.6 Hz, 2H), 3.75 (m, 4H,2×CH₂), 4.20 (dd, J=5.2, 12 Hz, 2H), 4.54 (m, 2H), 4.62 (m, 1H, CH),6.64 (d, J=6.4 Hz, 1H, OH). MS (m/z) 180.2 (M+1).

Step 2

1-Chloro-3-morpholin-4-yl-propan-2-ol (2.0 g, 11 mmol) was treated withthe solution of NH₃ in methanol (25% by weight, 20 mL) at roomtemperature. Nitrogen was bulbbed into the reaction mixture to removethe ammonia. Evaporation of solvent gave the hydrogen chloride salt of1-amino-3-morpholin-4-yl-propan-2-ol (2.0 g, 91%). ¹H NMR (DMSO-d₆) δ2.30 (d, J=6.0 Hz, 2H), 2.36 (m, 4H, NCH₂), 2.65 (dd, J=8.4, 12.8 Hz,1H), 2.91 (dd, J=3.6, 12.8 Hz, 1H), 3.52 (m, 4H, OCH₂), 3.87 (m, 1H,CH), 5.32 (s, 1H, OH), 8.02 (brs., 3H, NH₃ ⁺). MS (m/z) 161.1 (M+1).

Step 3

5-(5-Fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (120 mg, 0.4 mmol) was condensed with1-amino-3-morpholin-4-yl-propan-2-ol (74 mg, 0.48 mmol) to precipitate5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-hydroxy-3-morpholin-4-yl-propyl)-amide (65 mg, 36%). The motherliquid was evaporated to dryness and the residue was purified by flashchromatography to give additional 2N (70 mg, 39%). ¹H NMR (DMSO-d₆) δ2.28 (m, 1H), 2.32 (m, 1H), 2.40 (m, 4H), 2.40, 2.42 (2×s, 6H, 2×CH₃),3.15 (s, 1H), 3.31 (m, 1H), 3.55 (m, 4H), 3.78 (m, 1H), 4.73 (brs, 1H,OH), 6.82 (dd, J=4.5, 8.4 Hz, 1H), 6.90 (td, ²J=2.8, ³J=10.0 Hz, 1H),7.53 (m, 1H), 7.70 (s, 1H), 7.74 (dd, J=2.0, 9.6 Hz, 1H) (aromatic andvinyl), 10.87 (s, 1H, CONH), 13.66 (s, 1H, NH). LC-MS (m/z) 441.4 (M−1).

Synthesis of 2-hydroxy-7-oxa-4-azoniaspiro[3.5]nonane chloride

To a 1 L 3-neck round bottom flask, fitted with a thermocouple, nitrogeninlet and a 250 ml addition funnel, was charged morpholine (91.5 g, 91.5ml, 1.05 mole, 1.0 eq.) and 100 ml of ethanol. The solution was stirredrapidly while adding epichlorohydrin (100 g, 84.5 ml, 1.08 mole, 1.03eq.) from the addition funnel over about 30 minutes. The temperature wasmonitored and when the pot temperature reached 27° C., the reaction wascooled with an ice water bath. The clear solution was stirred for 18hours. The reaction was assayed by GC (dilute 5 drops of reactionmixture into 1 ml of ethanol and inject onto a 15 m DB-5 capillary GCcolumn with the following run parameters, Injector 250° C., detector250° C., initial oven temperature 28° C. warming to 250° C. at 10° C.per minute.) The reaction was complete with less than 3% morpholineremaining. The reaction was concentrated on the rotoevaporated at 50° C.with full house vacuum until no more distillate could be condensed. Theresulting oil was stored at room temperature for 24-48 hours or until asignificant mass of crystals was observed (seeded will speed up theprocess). The slurry was diluted with 250 ml of acetone and filtered.The solids were dried in the vacuum oven at 60° C. for 18-24 hours. Thisprovided 84 g of crystalline product. The mother liquors could beconcentrated and the crystallization process repeated in increaserecovery. ¹H NMR (400 MHz, DMSO-d₆) δ 6.55 (d, 1H), 4.64 (m, 1H), 4.53(m, 2H), 4.18 (m, 2H), 3.74 (m, 4H), 3.60 (m, 2H), 3.48 (m, 2H). ¹³C NMR(100 MHz, DMSO-d₆) δ 70.9, 61.39, 61.04, 60.25, 58.54, 57.80.

Synthesis of 1-amino-3-(4-morpholinyl)-2-propanol (Racemic)

To a 3 L 1-neck round bottom flask with a magnetic stir bas was charged2-hydroxy-7-oxa-4-azoniaspiro[3,5]nonane chloride (150 g, 835 mmole)followed by 23 wt. % anhydrous ammonia in methanol (2120 ml). The flaskwas stoppered and the resulting clear solution was stirred at 20-23° C.for 18 hours. GC under the conditions above showed no remaining startingmaterial. The stopper was removed and the ammonia allowed to bubble outof the solution for 30 minutes. The flask was then transferred to arotoevaporated and concentrated to a white solid with 45° C. bath andfull house vacuum. ¹H NMR (400 MHz, DMSO-d₆) δ 3.57 (dd, 2H), 3.3-3.5(m, 6H), 2.59 (m, 2H), 2.2-2.4 (m, 6H); ¹³C NMR (100 MHz DMSO-d₆) δ70.8, 67.1, 60.1, 53.8, 48.1.

Following the procedure described in Example 3 above but substituting2-(RS)-1-amino-3-morpholin-4-yl-propan-2-ol with2-(S)-1-amino-3-morpholin-4-yl-propan-2-ol prepared as described belowthe desired compound5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-(S)-hydroxy-3-morpholin-4-yl-propyl)-amide was obtained.

Synthesis of 1-amino-3-(4-morpholinyl)-2-propanol (Non-Racemic)

To 1 L 3-neck round bottom flask, fitted with mechanical stirring,thermocouple and addition funnel, was charged morpholine (91.5 g, 91.5ml, 1.05 mole, 1.0 eq.) and 45 ml of t-butanol. The solution was stirredrapidly while adding R-epichlorohydrin (100 g, 84.5 ml, 1.08 mole. 1.03eq.) from the addition funnel over about 30 minutes. The temperature wasmonitored and when the pot temperature reached 27° C., the reaction wascooled with an ice water bath. The clear solution was stirred for 18hours. The reaction was assayed by GC (dilute 5 drops of reactionmixture into 1 ml of ethanol and inject onto a 15 m DB-5 capillary GCcolumn with the following run parameters, Injector 250° C., detector250° C., initial oven temperature 28° C. warming to 250° C. at 10° C.per minute). The reaction was complete with less than 3% morpholineremaining. The solution was cooled to 10° C. and a 20 wt % solution ofpotassium t-butoxide in THF (576 g) was added dropwise keeping thetemperature less than 15° C. The resulting white slurry was stirred at10-15° C. for 2 hours and checked by GC using the above conditions. Noneof the chlorohydrin could be observed. The mixture was concentrated onthe rotoevaporated using 50° C. bath and full house vacuum. Theresulting mixture was diluted with water (500 ml) and methylenechloride. The phases were separated and the aqueous phase washed withmethylene chloride (500 ml). The combined organic layers were dried oversodium sulfate and concentrated to a clear, colorless oil. This provided145 g, 97% yield of the epoxide. ¹H NMR (400 MH_(z), DMSO-d₆) δ 3.3 (dd,4H), 3.1 (m, 1H), 2.6 (dd, 1H), 2.5 (dd, 1H), 2.4 (m, 4H), 2.2 (dd, 2H);¹³C NMR (100 MH_(z), DMSO-d₆) δ 65.4, 60.1, 53.1, 48.9, 43.4.

The above crude epoxide was charged to a 3 L 1-neck round bottom flaskwith a magnetic stir bar. Anhydrous ammonia in methanol (24% w/w 2.5 L)was added, the flask was stoppered and the mixture stirred at roomtemperature for 24 hours. GC under the conditions above showed noremaining starting material. The stopper was removed and the ammoniaallowed to bubble out of the solution for 30 minutes. The flask was thentransferred to a rotoevaporated and concentrated to a clear colorlessoil with 45° C. bath and full house vacuum. This provided 124 g ofproduct. ¹H NMR (400 MH_(z), DMSO-d₆) δ 3.57 (dd, 2H), 3.3-3.5 (m, 6H),2.59 (m, 2H), 2.2-2.4 (m, 6H); ¹³C NMR (100 MH_(z), DMSO-d₆) δ 70.8,67.1, 60.1, 53.8, 48.1.

Synthesis of 1-amino-3-(4-morpholinyl)-2-(S)-propanol

To 1 L 3-neck round bottom flask, fitted with mechanical stirring,thermocouple and addition funnel, was charged morpholine (91.5 g, 91.5ml, 1.05 mole, 1.0 eq.) and 200 ml of methanol. The solution was stirredrapidly while adding R-epichlorohydrin (100 g, 84.5 ml, 1.08 mole, 1.03eq.) from the addition funnel over about 30 minutes. The temperature wasmonitored and when the pot temperature reached 27° C., the reaction wascooled with an ice water bath. The clear solution was stirred for 18hours. The reaction was assayed by GC (dilute 5 drops of reactionmixture into 1 ml of ethanol and inject onto a 15 m DB-5 capillary GCcolumn with the following run parameters, Injector 250° C., detector250° C., initial oven temperature 28° C. warming to 250° C. at 10° C.per minute.) The reaction was complete with less than 3% morpholineremaining. The solution was cooled to 10° C. and a 25 wt. % solution ofsodium methoxide in methanol (233 g, 1.08 mole, 247 ml) was addeddropwise keeping the temperature less than 15° C. The resulting whiteslurry was stirred at 10-15° C. for 2 hours and checked by GC using theabove conditions. None of the chlorohydrin could be observed. Themixture was concentrated on the rotoevaporator using 50° C. bath andfull house vacuum. The resulting mixture was diluted with water (500 ml)and methylene chloride. The phases were separated and the aqueous phasewashed with methylene chloride (500 ml). The combined organic layerswere dried over sodium sulfate and concentrated to a clear, colorlessoil. This provided 145 g, 97% yield of1,2-epoxy-3-morpholin-4-ylpropane. ¹H NMR (400 MHz, DMSO-d₆) δ 3.3 (dd,4H), 3.1 (m, 1H), 2.6 (dd, 1H), 2.5 (dd, 1H), 2.4 (m, 4H), 2.2 (dd, 2H);¹³C NMR (100 MHz, DMSO-d₆) δ 65.4, 60.1, 53.1, 48.9, 43.4.

The above crude 1,2-epoxy-3-morpholin-4-ylpropane was charged to a 3 L1-neck round bottom flask with a magnetic stir bar. Anhydrous ammonia inmethanol (24% w/w 2.5 L) was added, the flask was stoppered and themixture stirred at room temperature for 24 hours. GC under theconditions above showed no remaining starting material. The stopper wasremoved and the ammonia allowed to bubble out of the solution for 30minutes. The flask was then transferred to a rotoevaporated andconcentrated to a clear colorless oil with 45° C. bath and full housevacuum. This provided 124 g of 1-amino-3-(4-morpholinyl)-2-(S)-propanol.¹H NMR (400 MHz, DMSO-d₆) δ 3.57 (dd, 2H), 3.3-3.5 (m, 6H), 2.59 (m,2H), 2.2-2.4 (m, 6H); ¹³C NMR (100 MHz, DMSO-d₆) δ 70.8, 67.1, 60.1,53.8, 48.1.

Imidazole amide (7.0 g, 32.3 mmol), amine (15.0 g, 64.6 mmol),5-fluorooxindole (4.93 g, 32.6 mmol), triethylamine (9.79 g, 96.9 mmol),and THF (88 ml) were mixed and heated to 60° C. A brown solution formed.After stirring for 24 h at 60° C., the yellow slurry was cooled to rt(room temperature) and filtered. The cake washed with 80 ml THF anddried overnight at 50° C. under house vacuum. A brown solid (23.2 g) wasobtained. The solid was slurried in 350 ml water for 5 h at rt andfiltered. The cake washed with 100 ml water and dried at 50° C. underhouse vacuum overnight. 8.31 g were obtained with 56% chemical yield.

A 0.25 L flask fitted with a thermometer, condenser, magnetic stirring,and nitrogen inlet was charged with 4.92 g 5-Fluorooxindole, 7.0 gImidazole amide, 15.5 g (R)-1-Amino-3-(4-morpholinyl)-2-propanol, 9.78 gTriethylamine and 88 ml Tetrahydrofuran. The mixture was heated to 60°C. for 16.5 hours. The reaction is cooled to ambient temperature andfiltered. The solids obtained are slurried (3) three successive times inacetonitrile at 11 ml/g, dried in vacuo for 3.6 g (25.25%). [HPLC,Hypersil BDS, C-18, 5μ, (6:4), Acetonitrile: 0.1M Ammonium Chloride,PHA-571437=4.05 min.] H¹NMR (DMSO): δ 10.86 (1H, bs); 7.75 (1H, d); 7.70(1H, s); 7.50 (1H, m); 6.88 (2H, m); 4.72 (1H, bs); 3.78 (1H, bs); 3.56(4H, m); 3.32 (6H, m); 3.15 (1H, m); 2.43 (8H, bm).

Example 4 Synthesis of2,4-dimethyl-5-[2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-1H-pyrrole-3-carboxylicacid (2-hydroxy-3-morpholin-4-yl-propyl)-amide

5-(2-Oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (113 mg, 0.4 mmol) was condensed with1-amino-3-morpholin-4-yl-propan-2-ol (74 mg, 0.48 mmol) to precipitate2,4-dimethyl-5-[2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-1H-pyrrole-3-carboxylicacid (2-hydroxy-3-morpholin-4-yl-propyl)-amide (77 mg, 45.3%). ¹H NMR(DMSO-d₆) δ 2.27 (m, 1H), 2.32 (m, 1H), 2.40 (m, 4H), 2.40, 2.42 (2×s,6H, 2×CH₃), 3.15 (s, 1H), 3.32 (m, 1H), 3.55 (m, 4H), 3.77 (m, 1H), 4.74(d, J=4.8 Hz, 1H, OH), 6.86 (d, J=7.6 Hz, 1H), 6.96 (t, J=7.2 Hz, 1H),7.10 (t, J=7.6 Hz, 1H), 7.49 (t, J=5.6 Hz, 1H), 7.61 (s, 1H), 7.77 (d,J=8.0 Hz, 1H) (aromatic and vinyl), 10.88 (s, 1H, CONH), 13.62 (s, 1H,NH). LC-MS (m/z) 425.4 (M+1).

Example 5 Synthesis of5-[5-chloro-2-oxo-1,2-dihydro-indol-(3Z)-ylidene-methyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-hydroxy-3-morpholin-4-yl-propyl)-amide

5-(5-Chloro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (126.6 mg, 0.4 mmol) was condensed with1-amino-3-morpholin-4-yl-propan-2-ol (74 mg, 0.48 mmol) to precipitate5-[5-Chloro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-hydroxy-3-morpholin-4-yl-propyl)-amide (107 mg, 58%). ¹H NMR(DMSO-d₆) δ 2.29 (m, 1H), 2.33 (m, 1H), 2.39 (m, 4H), 2.40, 2.42 (2×s,6H, 2×CH₃), 3.15 (s, 1H), 3.37 (m, 1H), 3.55 (m, 4H), 3.77 (m, 1H), 4.74(d, J=4.8 Hz, 1H, OH), 6.85 (d, J=8.4 Hz, 1H), 7.11 (dd, J=2.0, 8.0 Hz,1H), 7.53 (t, J=5.6 Hz, 1H), 7.75 (s, 1H), 7.97 (d, J=2.0 Hz, 1H)(aromatic and vinyl), 10.99 (s, 1H, CONH), 13.62 (s, 1H, NH). LC-MS(m/z) 457.4 (M−1).

The R and S stereoisomers can be prepared as follows.

Imidazole amide (7.0 g, 32.3 mmol), amine (15.5 g, 96.9 mmol),5-chlorooxindole (5.48 g, 32.6 mmol), triethylamine (14 ml), and THF (88ml) were mixed and heated to 60° C. A red solution formed. Afterstirring for 16 h at 60° C., the yellow slurry was cooled to rt andfiltered. The cake washed with 2×50 ml THF and dried overnight at 50° C.under house vacuum. 4.36 g were obtained with 29% chemical yield.

Imidazole amide (6.8 g, 31.3 mmol), amine (10.0 g, 62.5 mmol),5-chlorooxindole (5.3 g, 31.6 mmol), and THF (100 ml) were mixed andheated to 60° C. A red solution formed. After stirring for 68 h at 60°C., triethylamine (14 ml) was added and stirred for 5 h at 60° C.Reaction was not complete. Add 4.6 g of amine side chain, and stirredfor 20 h at 60° C. The yellow slurry was cooled to rt and filtered. Thecake washed with 2×50 ml THF and dried overnight at 50° C. under housevacuum. 5.48 g were obtained with 38% chemical yield.

Example 6 Synthesis of5-[5-bromo-2-oxo-1,2-dihydro-indol-(3Z)-ylidene-methyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-hydroxy-3-morpholin-4-yl-propyl)-amide

5-(5-Bromo-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (72.2 mg, 0.2 mmol) was condensed with1-amino-3-morpholin-4-yl-propan-2-ol (38 mg, 0.24 mmol) to precipitate5-[5-Bromo-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-hydroxy-3-morpholin-4-yl-propyl)-amide (55 mg, 55%). ¹H NMR(DMSO-d₆) δ 2.27 (m, 1H), 2.32 (m, 1H), 2.39 (m, 4H), 2.41, 2.42 (2×s,6H, 2×CH₃), 3.13 (s, 1H), 3.35 (m, 1H), 3.55 (m, 4H), 3.77 (m, 1H), 4.74(d, J=4.4 Hz, 1H, OH), 6.80 (d, J=8.4 Hz, 1H), 7.24 (dd, J=2.0, 8.0 Hz,1H), 7.51 (t, J=5.6 Hz, 1H), 7.76 (s, 1H), 8.09 (d, J=2.0 Hz, 1H)(aromatic and vinyl), 10.99 (s, 1H, CONH), 13.62 (s, 1H, NH). LC-MS(m/z) 503.4 (M−1).

Example 7 Synthesis of2,4-dimethyl-5-[2-oxo-1,2-dihydro-indol-(3Z)-ylidene-methyl]-1H-pyrrole-3-carboxylicacid (2-hydroxy-3-[1,2,3]triazol-1-yl-propyl)-amide

Step 1

A mixture of 3-[1,2,3]triazole (2.0 g, 29 mmol), epichlorohydrin (3.4ml, 43.5 mmol) and N,N-diisopropyl-ethylamine (2.6 mL, 15 mmol) inethanol (50 mL) was stirred at room temperature overnight. Afterremoving the solvents, the residue was purified by flash chromatography(CH₂Cl₂/CH₃OH=100/1-100/2-100/4) to give1-chloro-3-(1,2,3)-triazol-2-ylpropan-2-ol (2.1 g, 45%). ¹H NMR (CDCl₃)δ 3.52 (m, 2H, OH and CH₂), 3.60 (dd, J=5.2, 11.2 Hz, 1H), 4.36 (m, 1H,CH), 4.68 (m, 2H), 7.67 (s, 2H). MS (m/z) 162.1 (M+1) and1-chloro-3-(1,2,3)triazol-1-ylpropan-2-ol (2.3 g, 49%). ¹H NMR (CDCl₃) δ3.56 (s, 1H), 3.57 (s, 1H), 4.35 (m, 1H), 4.53 (dd, J=7.2, 14 Hz, 1H),4.67 (dd, J=3.8, 14 Hz, 1H), 7.67 (s, 1H), 7.71 (s, 1H). MS (m/z) 162.1(M+1).

Step 2

1-Chloro-3(1,2,3)triazol-1-ylpropan-2-ol (2.3 g, 13 mmol) was treatedwith the solution of NH₃ in methanol (25% by weight, 20 mL) at 60° C.overnight in a sealed pressure vessel. After cooling to roomtemperature, nitrogen was bulbbed into the reaction mixture to removethe ammonia. Evaporation of solvent gave the hydrogen chloride salt of1-amino-3-(1,2,3)triazol-1-ylpropan-2-ol (2.57 g, 100%). ¹H NMR(DMSO-d₆) δ 2.68 (dd, J=8.8, 12.8 Hz, 1H), 2.97 (dd, J=3.6, 12.8 Hz,1H), 4.15 (m, 1H), 4.44 (dd, J=6.4, 14 Hz, 1H), 4.57 (dd, J=4.6, 14 Hz,1H), 5.95 (d, J=5.2 Hz, 1H, OH), 7.77 (s, 1H), 8.01 (brs., 3H, NH₃ ⁺),8.12 (s, 1H). MS (m/z) 143.1 (M+1).

Step 3

5-(2-Oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (113 mg, 0.4 mmol) was condensed with1-amino-3(1,2,3)triazole-1-yl-propan-2-ol (85 mg, 0.48 mmol) toprecipitate2,4-dimethyl-5-[2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-1H-pyrrole-3-carboxylicacid (2-hydroxy-3-[1,2,3]triazol-1-yl-propyl)-amide (70 mg, 41%). ¹H NMR(DMSO-d₆) δ 2.45, 2.48 (2×s, 6H, 2×CH₃), 3.35 (m, 2H), 4.02 (m, 1H),4.32 (dd, J=7.6, 14 Hz, 1H), 4.53 (dd, J=3.4, 14 Hz, 1H), 5.43 (d, J=5.6Hz, 1H, OH), 6.91 (d, J=7.6 Hz, 1H), 7.01 (t, J=7.6 Hz, 1H), 7.15 (t,J=8.0 Hz, 1H), 7.66 (s, 1H), 7.12 (t, J=5.6 Hz, 1H), 7.74 (s, 1H), 7.77(d, J=7.6 Hz, 1H), 8.11 (s, 1H), 10.93 (s, 1H, CONH), 13.68 (s, 1H, NH).LC-MS (m/z) 405.4 (M−1).

Example 8 Synthesis of5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidene-methyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-hydroxy-3-[1,2,3]triazol-1-yl-propyl)-amide

5-(5-Fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (120 mg, 0.4 mmol) was condensed with 1-amino-3(1,2,3)triazol-1-yl-propan-2-ol (85 mg, 0.48 mmol) to precipitate5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-hydroxy-3-[1,2,3]triazol-1-yl-propyl)-amide (100 mg, 62%). ¹HNMR (DMSO-d₆) δ 2.42, 2.44 (2×s, 6H, 2×CH₃), 3.27 (m, 2H), 3.98 (m, 1H),4.27 (dd, J=7.6, 14 Hz, 1H), 4.50 (dd, J=3.4, 13.6 Hz, 1H), 5.38 (d,J=5.6 Hz, 1H, OH), 6.82 (dd, J=4.4, 8.4 Hz, 1H), 6.91 (td, ²J=2.4,³J=9.0 Hz, 1H), 7.70 (m, 3H), 7.75 (dd, J=2.4, 9.2 Hz, 1H), 8.11 (s.1H), 10.93 (s, 1H, CONH), 13.73 (s, 1H, NH). LC-MS (m/z) 423.4 (M−1).

Example 9 Synthesis of5-[5-chloro-2-oxo-1,2-dihydro-indol-(3Z)-ylidene-methyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-hydroxy-3-[1,2,3]triazol-1-yl-propyl)-amide

5-(5-Chloro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (126.6 mg, 0.4 mmol) was condensed with 1-amino-3(1,2,3)triazole-1-yl-propan-2-ol (85 mg, 0.48 mmol) to precipitate5-[5-Chloro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-hydroxy-3-[1,2,3]triazol-1-yl-propyl)-amide (48 mg, 27%). ¹H NMR(DMSO-d₆) δ 2.42, 2.44 (2×s, 6H, 2×CH₃), 3.27 (m, 2H), 3.99 (m, 1H),4.28 (dd, J=7.8, 14 Hz, 1H), 4.51 (dd, J=3.2, 14 Hz, 1H), 5.39 (d, J=6.0Hz, 1H, OH), 6.85 (d, J=8.4 Hz, 1H), 7.12 (dd, J=2.0, 8.2 Hz, 1H), 7.70(m, 2H), 7.74 (s, 1H), 7.97 (d, J=2.0 Hz, 1H), 8.07 (s, 1H), 10.99 (s,1H, CONH), 13.65 (s, 1H, NH). LC-MS (m/z) 439.4 (M−1).

Example 10 Synthesis of5-[5-bromo-2-oxo-1,2-dihydro-indol-(3Z)-ylidene-methyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-hydroxy-3-[1,2,3]triazol-1-yl-propyl)-amide

5-(5-Bromo-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (144.4 mg, 0.4 mmol) was condensed with1-amino-3(1,2,3)triazole-1-yl-propan-2-ol (85 mg, 0.48 mmol) toprecipitate5-[5-bromo-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-hydroxy-3-[1,2,3]triazol-1-yl-propyl)-amide (130 mg, 67%). ¹HNMR (DMSO-d₆) δ 2.41, 2.44 (2×s, 6H, 2×CH₃), 3.27 (m, 2H), 3.99 (m, 1H),4.28 (dd, J=7.6, 14 Hz, 1H), 4.50 (dd, J=3.6, 14 Hz, 1H), 5.40 (d, J=5.6Hz, 1H, OH), 6.81 (d, J=8.4 Hz, 1H), 7.24 (dd, J=2.0, 8.0 Hz, 1H), 7.70(m, 2H), 7.77 (s, 1H), 8.07 (s. 1H), 8.10 (d, J=1.6 Hz, 1H), 11.0 (s,1H, CONH), 13.64 (s, 1H, NH). LC-MS (m/z) 485.4 (M−1).

Synthesis of5-(5-bromo-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid,5-(5-chloro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid,5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid is described in Applicants' concurrently filed with the presentapplication on Feb. 14^(th), 2001, titled “PYRROLE SUBSTITUTED2-INDOLINONE—PROTEIN KINASE INHIBITORS”, Ser. No. 09/783,264, thedisclosure of which is incorporation herein in its entirety.

Example 11 Synthesis of 1-amino-3-(1,I-dioxo-λ⁶-thiomorpholin-4-yl)-propan-2-ol

To the solution of thiomorpholine (5.0 g, 48.7 mmol) in HOCH₃ (200 mL)was added the solution of oxone (36.0 g, 58.5 mmol) in H₂O (100 mL). Themixture was well stirred at 40° C. for 48 h and then cooled to 0° C.Aqueous NaOH was added dropwise to adjust pH=12. Solid was filtered outand washed with HOCH₃ (3×40 mL). The combined liquid was condensed andpurified by flash chromatograph on silicagel(CHCl₃/CH₃OH/NH₃.H₂O=3/1/0.1-2/1/0.1) to give thiomorpholine1,1-dioxide (6.2 g) in 93% yield. ¹H NMR (DMSO-d₆) δ 2.97 (m, 4H), 3.07(m, 4H), 3.42 (brs, 1H), MS (m/z) 136 (M+I).

The mixture of thiomorpholine 1,1-dioxide (2.5 g., 18.5 mmol) and(R)-(−)epichlorohydrin (1.55 mL, 20 mmol) in the mixture solventsethanol (50 mL) and H₂O (5 ml) was stirred at 25° C. for 24 h. Afterremoving solvent, the residue was purified by flash chromatography togive (R)-1-chloro-3-(I, 1-dioxo-λ⁶⁻-thiomorpholin-4-yl)-propan-2-ol (4.0g, 96%). ¹H NMR (DMSO-d₆) δ 2.50 (m, 2H), 2.94 (m, 4H), 305 (m, 4H),3.54 (dd, J=5.8, 11.2, Hz, 1H), 3.63 (dd, J=4.4, 11.2 Hz, 1H), 3.78 (m,H, CH), 5.10 (d, J=5.2 Hz, 1H, OH), MS (m/z) 228.2 (M+1).

(R)—I-Chloro-3-(1,1-dioxo-λ⁶-thiomorpholin-4-yl)-propan-2-ol (2.27 g, 10mmol) was treated with the solution of NH₃ in methanol (25% by weight,20 mL) at 50° C. for 12 h. After evaporation of solvents, the residuewas treated with anion exchange resin (AG1×8, OH form) in water to givecrude (S)-1-amino-3-(1,1-dioxo-λ⁶-thiomorpholin-4-yl), propan-2-ol (2.0g). It was contaminated by about 30% of its dimer and could barely bepurified by column chromatography. MS (m/z) 209.2 (M+1). Condensation of(S)-1-amino-3-(1,1-dioxo-λ⁶-thiomorpholin-4-yl)-propan-2-ol withoxindoles gave the desired indolinones (yield 50-80% afterpurification).

(R)-5-(2-Oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid [3-(1,1-dioxo-λ⁶-thiomorpholin-4-yl)-2-hydroxy-propyl]-amide

¹H NMR (DMSO-d₆) δ 2.39, 2.42 (2×s, 6H, 2×CH₃), 2.49 (m, 1H), 2.56 (m,1H), 2.97 (m, 4H), 3.07 (m, 4H), 3.16 (m, 1H), 3.34 (m, 1H), 3.74 (m,1H), 4.83 (d, J=4.8 Hz, 1H, OH), 6.86 (d, =7.6 Hz, 1H), 6.97 (t, J=7.4Hz, 1H), 7.11 (t, J=7.5 Hz, 1H), 7.50 (t, J=5.6 Hz, 1H), 7.61 (s, 1H),7.76 (d, J=7.6 Hz, 1H) (aromatic and vinyl), 10.88 (s, 1H, CONH), 13.62(s, 1H, NH), LC-MS (m/z) 473.4 (M+1).

(R)-5-(5-Fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxlicacid [3-(1,1-dioxo-λ₆-thiomorpholin-4-yl)-2-hydroxy-propyl]-amide

¹H NMR (DMSO-d₆) δ 2.40, 2.42 (2×s, 6H, 2×CH₃), 2.47 (m, 1H), 2.54 (m,1H), 2.97 (m, 4H), 3.06 (m, 4H), 3.17 (m, 1H), 3.30 (m, 1H), 3.74 (m,1H), 4.83 (d, J=4.4 Hz, 1H), 6.82 (t, J=4.0 Hz, 1H) 6.91 (td, ²J=2.8,³J=9.0 Hz, 1H), 7.53 (t, J=5.8 Hz, 1H), 7.70 (s, 1H) 7.75 (dd, J=2.4,9.2 Hz, 1H), 10.88 (s, 1H), 13.67 (s, 1H). LC-MS (m/z) 491.4 (M+1).

(R)-5-(5-Chloro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid [3-(1,1-dioxo-λ⁶-thiomorpholin-4-yl)-2-hydroxy-propyl]-amide

¹H NMR (DMSO-d₆) δ 2.40, 2.42 (2×s, 6H, 2×CH₃), 2.45 (m, 1H), 2.53 (m,1H), 2.96 (m, 4H), 3.06 (m, 4H), 3.17 (m, 1H), 3.33 (m, 1H), 3.75 (m,1H), 4.83 (d, J=4.4 Hz, 1H, OH), 6.85 (t, J=8.4 Hz, 1H), 7.11 (dd,J=2.2, 8.2 Hz, 1H), 7.53 (t, J=5.5 Hz, 1H), 7.75 (s, 1H), 7.97 (d, J=2.0Hz, 1H), 10.98 (s, 1H), 13.62 (s, 1H), LC-MS (m/z) 507.2 (M+1).

(R)-5-(5-Bromo-2-oxo-1,2-dihydro-indol-3-ylidenemethyl),2,4-dimethyl-1H-pyrrole-3-carboxylic acid[3-(1,1-dioxo-λ⁶-thiomorpholin-4-yl)-2-hydroxy-propyl]-amide

¹H NMR (DMSO-d₆) δ 2.41, 2.42 (2×s, 6H, 2×CH₃), 2.47 (m, 1H), 2.54 (m,1H), 2.97 (m, 4H), 3.06 (m, 4H), 3.18 (m, 1H), 3.30 (m, 1H), 3.74 (m,1H), 4.83 (d, J=4.8 Hz, 1H), 6.81 (d, J=8.4 Hz, 1H), 7.24 (dd, J=1.8,8.2 Hz, 1H), 7.53 (t, J=5.8, 1H), 7.76 (s, 1H), 8.09 (d, J=2.0 Hz, 1H),10.98 (s, 1H) 13.62 (s, 1H), LC-MS (m/z) 553.6 (M+1).

Example 12 Synthesis of (S) or(R)-1-methylamino-3-morpholin-4-yl-propan-2-ol

The mixture of morpholine (1.74 mL, 20 mmol) and (R)-epichlorohydrin(1.56 mL, 20 mmol) in ethanol (10 mL) was stirred at r.t. for 48 h.Alter removing the solvent, the residue was treated with the solution ofCH₃NH₂ in water (40% by weight, 20 mL) at r.t. for 14 h. Removal of thesolvents gave the crude (S)-1-methylamino-3-morpholin-4-yl-propan-2-ol,which could be purified by vacuum distillation or column chromatography(2.4 g. 70%). (R)-1-Methylamino-3-morpholin-4-yl-propan-2-ol was madefrom (S)-epichlorohydrin in 76% yield. ¹H NMR (CDCl₃) δ 2.33 (dd, J=3.6,12.4 Hz, 1H), 2.42 (m, 1H), 2.44 (dd, J=2.8, 9.8 Hz, 2H), 2.45 (s, 3H),2.53 (dd, J=7.6, 11.8 Hz, 1H), 2.62 (m, 2H), 2.65 (dd, J=3.6, 12.0 Hz,1H), 3.71 (m, 4H), 3.85 (m, 1H), ¹³CNMR (CDCl₃) δ 67.06, 65.58, 62.80,55.87, 53.94, 36.72, MS (m/z) 175 (M+1).

(S)-1-Methylamino-3-morpholin-4-yl-propan-2-ol condensed with5-fluoroxindole furnished(R)-5-(5-Fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-hydroxy-3-morpholin-4-yl-propyl)-methyl-amide

¹H NMR (DMSO-d₆) δ 2.0 (s, 3H), 2.15 (m, 1H), 2.25 (m, 6H), 2.28 (m,2H), 2.42 (s, 1H), 2.95 (s, 1H), 3.0 (s, 3H), 3.25 (m, 2H), 3.57 (m,2H), 3.97 & 3.68 (2×brs, 1H), 4.80 & 4.74 (2×brs, 1H), 6.82 (dd, J=4.0,8.0 Hz, 1H),

6.90 (td, ²J=2.3, ³J=8.7 Hz, 1H), 7.67 (s, 1H), 7.71 (dd, J=2.2, 9.0 Hz,1H), 10.86 (s, 1H), 13.57 (s, 1H), LC-MS (m/z) 457.2 (M+1).

(R)-1-amino-3 morpholin-4-yl-propan-2-ol furnished(S)-5-(5-Fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-hydroxy-3-morpholin-4-yl-propyl)-methyl-amide

¹H NMR (DMSO-d₆) δ 2.0 (m, 3H), 2.10 (m, 1H), 2.22 (m, 6H), 2.25 (m,2H), 2.38 (m, 1H), 2.90 (s, 1H), 2.96 (s, 3H), 3.27 (m, 2H), 3.52 (m,2H), 3.93 & 3.64 (2×brs, 1H), 4.75 & 4.70(2×brs, 1H), 6.77 (dd, J=4.6,8.2 Hz, 1H), 6.85 (td, ²J=2.5, ³J=8.8 Hz, 1H), 7.62 (s, 1H), 767 (dd,J=2.0, 9.6 Hz, 1H), 10.81 (s, 1H) 13.52 (s, 1H), LC-MS (m/z) 457.2(M+1).

Example 13

Amine Side-Chain Preparation

3-(1-H-tetrazol-1-yl)]-2-hydroxy-1-chloropropane and3-(2-H-tetrazol-2-yl)-2-hydroxy-1-chloropropane

6.905 g of tetrazole (100 mmol) and 1.75 ml of diisopropylethylamine (10mmol) and 11.73 ml of epichlorohydrine (150 mmol) in anhydrousacetonitrile (30 ml) was stirred at 60° C. for 4 hours. The obtainedsolution was evaporated, dried on highvac and purified on a column ofsilica in chloroform-methanol 100:8. The first fraction provided pure3-(2-H-tetrazol-2-yl)-2-hydroxy-1-chloropropane, 6.215 g (colorless oil,38% Y), the second fraction yielded 9.208 g of pure3-(1-H-tetrazol-1-yl)]-2-hydroxyl-1-chloropropane (sticky gum; 57% Y).

3-(1-H-tetrazol-1-yl)]-2-hydroxy-1-aminopropane

9.110 g of 3-(1-H-tetrazol-1-yl)]-2-hydroxy-1-chloropropane, 15 g ofpotassium carbonate and 130 ml of saturated methanolic ammonia wasstirred for 21 hours, then filtered and evaporated. The residue purifiedon a column of silica in chloroform-methanol-aqueous ammonia 80:35:4.Y=7.326 g of a white sticky gum (91.5% th).

3-(2-H-tetrazol-2-yl)]-2-hydroxy-1-aminopropane

6.105 g of 3-(2-H-tetrazol-2-yl)]-2-hydroxy-1-chloropropane, 10 g ofpotassium carbonate and 95 ml of saturated methanolic ammonia wasstirred for 21 hours, then filtered and evaporated. The residue purifiedon a column of silica in chloroform-methanol-aqueous ammonia 60:25:2.Y=3.726 g of a white crystalline solid (69.5% th).

3-[(2R,6S)-2,6-dimethylmorpholin-4-yl]-2-hydroxy-1-aminopropane

4.7 ml of epichlorohydrine (60 mmol) was added to an ice-cooled solutionof cis-2,6-dimethylmorpholine (4.607 g, 40 mmol) in trifluoroethanol (5ml). The solution was stirred at 0-5° C. for 1 hr., the cooling bathremoved and stirred at RT for additional 5 hrs. The mixture wasevaporated on highvac, the obtained oily residue was dissolved inanhydrous ethanol (50 ml), the solution was cooled on ice bath, solidsodium methoxide (2.27 g) was added in two portions and the mixture wasstirred at 0-5° C. for 2 hrs. Reaction mixture was then filtered, saltswashed with ethanol (30 ml) and combined filtrates added to ice-cooledconcentrated aqueous ammonia (200 ml). The mixture was stirred at RT for12 hrs, then evaporated on highvac. The residue was purified on a columnof silica in mixture chloroform-methanol-7M methanolic ammonia 80:15:3.Y=5.75 g of a white crystalline hygroscopic solid (76.3% th).

(2S)-3-(3-methyl-2,5-dioxoimidazolidin-1-yl)-2-hydroxy-1-chloropropaneand(2S)-3-(3-methyl-2,5-dioxoimidazolidin-1-yl)-2-hydroxy-1-aminopropropane

3.423 g of 3-methyl-2,5-dioxoimidazolidine (3.423 g) and 3.60 ml of Repichlorophydrine (−) (99% e.e.) and 0.30 ml of Barton base (1.5 mmol)in anhydrous acetonitrile was stirred at 60° C. for 20 hrs. The obtainedsolution was evaporated on highvac and purified on a column of silica ina mixture of chloroform-methanol (a gradient 5 to 20% of methanol) toobtain 5.572 g of the chloro-compound as a white amorphous solid (90%Y). The chloride was transformed into amine as follows. The obtainedhydroxy-chloro intermediate was dissolved in methanolic ammonia(saturated with ammonia gas), potassium carbonate was added and themixture was stirred in closed flask for 2½ days. The reaction mixturewas filtered, filtrates evaporated. The residue was purified on a silicacolumn in a mixture chloroform-methanol-conc. aqueous ammonia 80:15:1.5.

Example 145-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[2-hydroxy-3-(2H-tetraazol-2-yl)propyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide(Compound 22) (general procedure)

72 mg of 3-(2-H-tetrazol-2-yl)]-2-hydroxy-1-aminopropane was added tothe slurry of 105 mg (0.25 mmol) of5-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid 1-oxy-7-azabenztriazole ester [prepared by activating(3Z)-3-({3,3-dimethyl-4-carboxy-1-H-pyrrol-2-yl}methylene)-5-fluoro-1,3-dihydro-2H-indol-2-one(480 mg; 1.6 mmol) with the HATU reagent (570 mg; 1.5 mmol) in thepresence of Hunig base (3.0 mol; 0.525 ml) in DMF (5 ml) and isolated inpure form by precipitation with chloroform (5 ml) and drying on highvacuum in 92% yield (579 mg)] in anhydrous dimethylacetamide (1.5 ml).The mixture was stirred for 30 min and evaporated on highvac. Theresidue was suspended in a mixture methanol-diethylamine 20:1 (3 ml),allowed to crystallize in the refrigerator (5° C.) for 1 hr, filtered,the precipitate washed with ice-cold methanol and dried on highvac.Y=106 mg of an orange crystalline solid.

Example 155-[(Z)-(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[2-hydroxy-3-(2H-tetraazol-2-yl)propyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide(Compound 23)(general procedure)

72 mg of 3-(2-H-tetrazol-2-yl)]-2-hydroxy-1-aminopropane was added tothe slurry of 109 mg (0.25 mmol) of5-[(Z)-(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid 1-oxy-7-azabenztriazole ester [prepared by activating(3Z)-3-([3,3-dimethyl-4-carboxy-1-H-pyrrol-2-yl)methylene)-5-chloro-1,3-dihydro-2H-indol-2-one(1.520 g; 4.8 mmol) with the HATU reagent (1.768 g; 4.65 mmol) in thepresence of Hunig base (9.0 mmol; 1.58 ml) in DMF (20 ml) and isolatedin pure form by precipitation with chloroform (20 ml) and drying on highvacuum in 94% yield (1.907 g)] in anhydrous dimethylacetamide (1.5 ml).The mixture was stirred for 30 min and evaporated on highvac. Theresidue was suspended in a mixture methanol-diethylamine 20:1 (3 ml),allowed to crystallize in the refrigerator (5° C.) for 1 hr, filtered,the precipitate washed with ice-cold methanol and dried on highvac.Y=109 mg of an orange crystalline solid.

Example 16N-[2-hydroxy-3-(2H-tetraazol-2-yl)propyl]-2,4-dimethyl-5-[(Z)-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]methyl)-1H-pyrrole-3-carboxamide(Compound 24) (general procedure)

72 mg of 3-(2-H-tetraazol-2-yl)]-2-hydroxy-1-aminopropane was added tothe slurry of 121.5 mg (0.25 mmol) of5-[(Z)-(5-trifluoromethoxy-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid 1-oxy-7-azabenztriazole ester [prepared by activating(3Z)-3-((3,3-dimethyl-4-carboxy-1-H-pyrrol-2-yl)methylene)-5-trifluoromethoxy-1,3-dihydro-2H-indol-2-one(1.768 g; 4.8 mmol) with the HATU reagent (1.758 g; 4.8 mmol) in thepresence of Hunig base (9.0 mmol; 1.58 ml) in DMF (25 ml) and isolatedin pure form by evaporation and precipitation with anhydrousacetonitrile and drying on high vacuum in 85.5% yield (1.929 g)] inanhydrous dimethylacetamide (1.5 ml). The mixture was stirred for 30 minand evaporated on highvac. The residue was suspended in a mixturemethanol-diethylamine 20:1 (3 ml), allowed to crystallize in therefrigerator (5° C.) for 1 hr, filtered, the precipitate washed withice-cold methanol and dried on highvac. Y=113 mg of an orangecrystalline solid.

Example 175-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[2-hydroxy-3-(1H-tetraazol-1-yl)propyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide(Compound 25)

This was prepared according to the procedure of Example 14 from 72 mg ofthe corresponding amine. Y=113 mg of an orange crystalline solid.

Example 185-[(Z)-(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[2-hydroxy-3-(1H-tetraazol-1-yl)propyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide(Compound 26)

This was prepared according to the procedure of Example 15 from 72 mg ofthe corresponding amine. Y=122 mg of an orange crystalline solid.

Example 19N-[2-hydroxy-3-(1H-tetraazol-1-yl)propyl]-2,4-dimethyl-5-[(Z)-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]methyl]-1H-pyrrole-3-carboxamide(Compound 27)

This was prepared according to the procedure of Example 16 from 72 mg ofthe corresponding amine. Y=118 mg of an orange crystalline solid.

Example 20N-[3-[(2R,6S)-2,6-dimethylmorpholin-4-yl]-2-hydroxypropyl)-5-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide(Compound 28)

This was prepared according to the procedure of Example 14 from 95 mg ofthe corresponding amine. Y=99 mg of an orange crystalline solid.

Example 215-[(Z)-(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[3-[(2R,6S)-2,6-dimethylmorpholin-4-yl]-2-hydroxypropyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide(Compound 29)

This was prepared according to the procedure of Example 15 from 95 mg ofthe corresponding amine. Y=101 mg of an orange crystalline solid.

Example 22 N-[3-[(2R,6S)-2,6-dimethylmorpholin-4-yl]-2-hydroxypropyl]-2,4-dimethyl-5-[(Z)-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]methyl]-1H-pyrrole-3-carboxamide(Compound 30)

This was prepared according to the procedure of Example 16 from 95 mg ofthe corresponding amine. Y=89 mg of an orange crystalline solid.

Example 235-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[(2S)-2-hydroxy-3-(3-methyl-2,5-dioxoimidazolidin-1-yl)]2,4-dimethyl-1H-pyrrole-3-carboxamide(Compound 34)

This was prepared according to the procedure of Example 14 from 95 mg ofthe corresponding amine. Y=109 mg of an orange crystalline solid.

Example 245-[(Z)-(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[2S)-2-hydroxy-3-(3-methyl-2,5-dioxoimidazolidin-1-yl)propyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide(Compound 36)

This was prepared according to the procedure of Example 15 from 95 mg ofthe corresponding amine. Y=107 mg of an orange crystalline solid.

Example 25N-[(2S)-2-hydroxy-3-(3-methyl-2,5-dioxoimidazolidin-1-yl)propyl]-2,4-dimethyl-5-{(Z)-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]methyl}-1H-pyrrole-3-carboxamide(Compound 35)

This was prepared according to the procedure of Example 16 from 95 mg ofthe corresponding amine. Y=123 mg of an orange crystalline solid.

Example 265-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[(2R)-2-hydroxy-3-(3-methyl-2,5-dioxoimidazolidin-1-yl)propyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide(Compound 31)

This was prepared according to the procedure of Example 14 from 95 mg ofthe corresponding amine. Y=1110 mg of an orange crystalline solid.

Example 275-[(Z)-(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[(2R)-2-hydroxy-3-(3-methyl-2,5-dioxoimidazolidin-1-yl)propyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide(Compound 32)

This was prepared according to the procedure of Example 15 from 95 mg ofthe corresponding amine. Y=103 mg of an orange crystalline solid.

Example 28N-[(2R)-2-hydroxy-3-(3-methyl-2,5-dioxolmidazolidin-1-yl)propyl]-2,4-dimethyl-5-{(Z)-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidenelmethyl}-1H-pyrrole-3-carboxamide(Compound 33)

This was prepared according to the procedure of Example 16 from 95 mg ofthe corresponding amine. Y=120 mg of an orange crystalline solid.

Example 29 5-Formyl-2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid ethylester

DMF (4 mL, 3 eq) was cooled with stirring in an ice bath. To this wasadded POCL₃ (1.1 eq., 1.8 mL). After 30 minutes, a solution of the3,5-dimethyl-4-ethylester pyrrole (4 g, 17.4 mmol) in DMF (2M, 9 mL) wasadded to the reaction and stirring continued. After 10 min, the reactionmixture solidified. This was diluted with 5 mL DMF and heated in 90° C.oil bath. After 1 hr, the reaction was cooled to room temperature anddiluted with water (100 mL) and basified to pH=11 with 1 N NaOH. Theproduct was extracted into methylene chloride (3×200 mL) and the organiclayers were washed with brine (200 mL), dried (MgSO4) and concentratedto afford 4.3 g (95%) of5-formyl-2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid ethyl ester as abrown solid. ¹H NMR (360 MHz, DMSO-d6) δ 12.5 (brs, 1H, NH), 9.11 (s,1H, CHO), 7.35 (s, 5H, ArH), 3.98 (q, J=6.8 and 7.2 Hz, 2H, OCH ₂CH₃),2.48 (s, 3H, CH ₃), 0.98 (t, J=7 Hz, 3H, OCH₂CH ₃).

5-Formyl-2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid

5-Formyl-2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid ethyl ester wasdissolved in water (100 mL) and methanol (45 mL) with stirring. AddedKOH (2 eq. 1.9 g) and heated in 100° C. After 2.5 h, cooled to roomtemperature and the remaining ester was removed by extracting into ethylacetate (200 mL), dried and concentrated. The water layer was acidifiedto pH=3 using 2N HCl. The white solid was removed by filtration, rinsingwith water. The solid was re-concentrated from toluene, triturated withhexanes and dried to afford 2 g (52%) of an off-white solid. ¹H NMR (360MHz, DMSO-d6) δ 12.46 (br s, 1H, CO₂H), 11.95 (s, 1H, NH), 9.08 (s, 1H,CHO), 7.36 (s, 5H, ArH), 2.49 (s, 3H, CH ₃). MS m/z (relative intensity%, ion) found 229 (100, M⁺); calc. 229.2.

5-Formyl-2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid(3-diethylamino-2-hydroxy-propyl)-amide

A mixture of 5-Formyl-2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid(1.0 gm, 4.36 mmol), 1-amino-3-diethylamino-2-propanol (950 mg, 6.54mmol), DDC (900 mg, 4.36 mmol) and HOBt (884 mg, 6.54 mmol) inchloroform (60 mL) was stirred at rt for 12 hrs. The reaction was pouredinto sat. Sodium bicarbonate (60 mL) and to it was added 1N NaOH (8 mL).It was then extracted with EtOAc (3×100 mL), washed with water andbrine, dried and concentrated to give 400 mg of5-Formyl-2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid(3-diethylamino-2-hydroxy-propyl)-amide.

Example 30 Procedure for the Synthesis of Compounds 11-21

A 0.36 M solution of each oxindole is prepared in DMSO as well as a0.576 M solution of each aldehyde. 300 uL of the appropriate oxindole ismixed with 300 uL of the appropriate aldehyde in the presence of 200 uLof DMSO. Then 40 mg of the Diethylenetriamine scavenger resin is added.The mixture is placed in a Robbins Block and the block is sealed andplaced into a 60° C. oven where it will shake for 18 hours.

After 18 hours, the Robbins Block is removed from the oven. The top sealof the block is removed and 800 uL of DMSO is added to the mixture. Thenthe block is resealed and again placed in the 60° C. oven where itrotates continuously for 1 hour.

After the 1 hour is complete, the Robbins Block is removed from the ovenand allowed to cool. The bottom seal of the Robbins Block is carefullyremoved and the entire block is fitted into the filtration device, whichenables the newly synthesized compounds to be filtered away from theresin.

Example 31 3-[1-H-(7-azabenztriazolyl)-oxy]-2-hydroxy-1-aminopropane

4.083 g of 1-hydroxy-7-azabenztriazole (30 mmol) and 0.53 ml ofdiisopropylamine (3 mmol) and 4.70 ml of epichlorohydrine in anhydrouschloroform was stirred at 60° C. for 2 hours. The reaction mixture waspoured onto a column of silica and eluted with a mixturechloroform-methanol 100:3. The obtained hydroxy-chloro intermediate(4.83 g, pale yellow oil, 70% Y) was dissolved in methanolic ammonia(100 ml, saturated with ammonia gas), 8.3 g of potassium carbonate wasadded and the mixture was stirred in closed flask for 2½ days. Thereaction mixture was filtered, filtrates evaporated. The residue waspurified on a silica column in a mixture chloroform-methanol-conc.aqueous ammonia 80:15:1.5 Y=2.793 g of a white crystalline solid (63%th. from the chloride)

3-[1-H-(benztriazolyl)-oxy]-2-hydroxy-1-chloropropane and3-[1-H-(benztriazolyl-3-N-oxido)]-2-hydroxy-1-chloropropane

12.162 g of hydroxyazabenztriazole (90 mmol), 1.59 ml ofdiisopropylethylamine (9 mmol) and 14.1 ml of epichlorohydrine (180mmol) in anh. Chloroform was stirred at 55° C. for 2 hours. The reactionmixture was evaporated, the residue was dried on highvac, then purifiedon a silica column in a mixture chloroform-methanol 100:5. The firstfractions provided 3-[1-H-(benztriazolyl)-oxy]-2-hydroxy-1-chloropropane10.570 g (pale yellow honey; 51.5% Y), followed by fraction of3-[1-H-(benztriazolyl-3-N-oxido)]-2-hydroxy-1-chloropropane 9.990 g(white crystalline solid, 48.5% Y)

3-[1-H-(benztriazolyl-3-N-oxido)]-2-hydroxy-1-aminopropane

Was prepared by aminolysis of3-[1-H-(benztriazolyl-3-N-oxido)]-2-hydroxy-1-chloropropane in analogyto the synthesis of3-[1-H-(7-azabenztriazolyl)-oxy]-2-hydroxy-1-aminopropane

Example 325-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[2-hydroxy-3-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)propyl]-2,4-dimenthyl-1H-pyrrole-3-carboxamide(general procedure)

105 mg of 3-[1-H-(7-azabenztriazolyl)-oxy]-2-hydroxy-1-aminopropane wasadded to the slurry of 105 mg (0.25 mmol) of5-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimenthyl-1H-pyrrole-3-carboxylicacid 1-oxy-7-azabenztriazole ester [prepared by activating(3Z)-3-({3,3-dimethyl-4-carboxy-1-H-pyrrol-2-yl}methylene)-5-fluoro-1,3-dihydro-2H-indol-2-one(480 mg; 1.6 mmol) mmol; 0.525 ml) in DMF (5 ml) and isolated in pureform by precipitation with chloroform (5 ml) and drying on high vacuumin 92% yield (579 mg)] in anhydrous dimethylacetamide (1.5 ml). Themixture was stirred for 30 min and evaporated on highvac. The residuewas suspended in a mixture methanol-diethylamine 20:1 (3 ml), allowed tocrystallize in the refrigerator (5° C.) for 1 hr, filtered, theprecipitate washed with ice-cold methanol and dried on highvac. Y=121 mgof an orange crystalline solid.

Example 335-[(Z)-(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[2-hydroxy-3-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)propyl]-2,4-dimenthyl-1H-pyrrole-3-carboxamide(general procedure)

105 mg of 3-[1-H-(7-azabenztriazolyl)-oxy]-2-hydroxy-1-aminopropane wasadded to the slurry of 109 mg (0.25 mmol) of5-[(Z)-(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid 1-oxy-7-azabenztriazole ester [prepared by activating(3Z)-3-({3,3-dimethyl-4-carboxy-1-H-pyrrol-2-yl}methylene)-5-chloro-1,3-dihydro-2H-indol-2-one(1.520 g; 4.8 mmol) with the HATU reagent (1.768 g; 4.65 mmol) in thepresence of Hunig base (9.0 mmol; 1.58 ml) in DMF (20 ml) and isolatedin pure form by precipitation with chloroform (20 ml) and drying on highvacuum in 94% yield (1.907 g)] in anhydrous dimethylacetamide (1.5 ml).The mixture was stirred for 30 min and evaporated on highvac. Theresidue was suspended in a mixture methanol-diethylamine 20:1 (3 ml),allowed to crystallize in the refrigerator (5° C.) for 1 hr, filtered,the precipitate washed with ice-cold methanol and dried on highvac.Y=130 mg of an orange crystalline solid.

Example 345-[(Z)-(5-trifluoromethoxy-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[2-hydroxy-3-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)propyl]-2,4-dimenthyl-1H-pyrrole-3-carboxamide(general procedure)

105 mg of 3-[1-H-(7-azabenztriazolyl)-oxy]-2-hydroxy-1-aminopropane wasadded to the slurry of 121.5 mg (0.25 mmol) of5-[(Z)-(5-trifluoromethoxy-2-oxo1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid 1-oxy-7-azabenztriazole ester [prepared by activating(3Z)-3-({3,3-dimethyl-4-carboxy-1-H-pyrrol-2-yl}methylene)-5-trifluoromethoxy-1,3-dihydro-2H-indol-2-one(1.768 g; 4.8 mmol) with the HATU reagent (1.758 g; 4.8 mmol) in thepresence of Hunig base (9.0 mmol; 1.58 ml) in DMF (25 ml) and isolatedin pure form by evaporation and precipitation with anhydrousacetonitrile and drying on high vacuum in 85.5% yield (1.929 g)] inanhydrous dimethylacetamide (1.5 ml). The mixture was stirred for 30 minand evaporated on highvac. The residue was suspended in a mixturemethanol-diethylamine 20:1 (3 ml), allowed to crystallize in therefrigerator (5° C.) for 1 hr, filtered, the precipitate washed withice-cold methanol and dried on highvac. Y=142 mg of an orangecrystalline solid.

Example 355-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[2-hydroxy-3-(3-oxido-1H-1,2,3-benzotriazol-1-yl)propyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide

This was prepared according to the procedure of Example 32 from 105 mgof the corresponding amine. Y=120 mg of an orange crystalline solid.

Example 365-[(Z)-(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[2-hydroxy-3-(3-oxido-1H-1,2,3-benzotriazol-1-yl)propyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide

This was prepared according to the procedure of Example 33 from 105 mgof the corresponding amine. Y=127 mg of an orange crystalline solid.

Example 37N-[2-hydroxy-3-(3-oxido-1H-1,2,3-benzotriazol-1-yl)propyl]-2,4-dimethyl-5-[(Z)-[2-oxo-5-(trifluoromethoxy)-1,2-dihydro-3H-indol-3-ylidene]methyl)-1H-pyrrole-3-carboxamide

This was prepared according to the procedure of Example 34 from 105 mgof the corresponding amine. Y=141 mg of an orange crystalline solid.

Biological Examples

The following assays are employed to find those compounds demonstratingthe optimal degree of the desired activity.

A. Assay Procedures.

The following assays may be used to determine the level of activity andeffect of the different compounds of the present invention on one ormore of the PKs. Similar assays can be designed along the same lines forany PK using techniques well known in the art.

Several of the assays described herein are performed in an ELISA(Enzyme-Linked Immunosorbent Sandwich Assay) format (Voller, et al.,1980, “Enzyme-Linked Immunosorbent Assay,” Manual of ClinicalImmunology, 2d ed., Rose and Friedman, Am. Soc. Of Microbiology,Washington, D.C., pp. 359-371). The general procedure is as follows: acompound is introduced to cells expressing the test kinase, eithernaturally or recombinantly, for a selected period of time after which,if the test kinase is a receptor, a ligand known to activate thereceptor is added. The cells are lysed and the lysate is transferred tothe wells of an ELISA plate previously coated with a specific antibodyrecognizing the substrate of the enzymatic phosphorylation reaction.Non-substrate components of the cell lysate are washed away and theamount of phosphorylation on the substrate is detected with an antibodyspecifically recognizing phosphotyrosine compared with control cellsthat were not contacted with a test compound.

The presently preferred protocols for conducting the ELISA experimentsfor specific PKs is provided below. However, adaptation of theseprotocols for determining the activity of compounds against other RTKs,as well as for CTKs and STKs, is well within the scope of knowledge ofthose skilled in the art.

Other assays described herein measure the amount of DNA made in responseto activation of a test kinase, which is a general measure of aproliferative response. The general procedure for this assay is asfollows: a compound is introduced to cells expressing the test kinase,either naturally or recombinantly, for a selected period of time afterwhich, if the test kinase is a receptor, a ligand known to activate thereceptor is added. After incubation at least overnight, a DNA labelingreagent such as 5-bromodeoxyuridine (BrdU) or H³-thymidine is added. Theamount of labeled DNA is detected with either an anti-BrdU antibody orby measuring radioactivity and is compared to control cells notcontacted with a test compound.

GST-Flk-1 Bioassay

This assay analyzes the tyrosine kinase activity of GST-Flk 1 onpoly(glu,tyr) peptides.

Materials and Reagents:

-   -   1. Corning 96-well ELISA plates (Corning Catalog No. 5805-96).    -   2. poly(glu,tyr) 4:1, lyophilizate (Sigma Catalog #P0275).    -   3. Preparation of poly(glu,tyr)(pEY) coated assay plates: Coat 2        ug/well of poly(glu,tyr)(pEY) in 100 ul PBS, hold at room        temperature for 2 hours or at 4° C. overnight. Cover plates well        to    -    prevent evaporation.    -   4. PBS Buffer: for 1 L, mix 0.2 g KH₂PO₄, 1.15 g Na₂HPO₄, 0.2 g        KCl and 8 g NaCl in approx. 900 ml dH₂O. When all reagents have        dissolved, adjust the pH to 7.2 with HCl. Bring total    -    volume to 1 L with dH₂O.    -   5. PBST Buffer: to 1 L of PBS Buffer, add 1.0 ml Tween-20.    -   6. TBB—Blocking Buffer: for 1 L, mix 1.21 g TRIS, 8.77 g NaCl, 1        ml TWEEN-20 in

 approximately 900 ml dH₂O. Adjust pH to 7.2 with HCl. Add 10 g BSA,stir to dissolve. Bring

 total volume to 1 L with dH₂O. Filter to remove particulate matter.

-   -   7. 1% BSA in PBS: To make a 1× working solution, add 10 g BSA to        approx. 990 ml PBS buffer,

 stir to dissolve. Adjust total volume to 1 L with PBS buffer, filter toremove particulate matter.

-   -   8. 50 mM Hepes pH 7.5.    -   9. GST-Flk1cd purified from sf9 recombinant baculovirus        transformation (SUGEN, Inc.).    -   10. 4% DMSO in dH₂O.    -   11. 10 mM ATP in dH₂O.    -   12. 40 mM MnCl₂    -   13. Kinase Dilution Buffer (KDB): mix 10 ml Hepes (pH 7.5), 1 ml        5M NaCl, 40 μL 100 mM

 sodium orthovanadate and 0.4 ml of 5% BSA in dH₂O with 88.56 ml dH₂O.

-   -   14. NUNC 96-well V bottom polypropylene plates, Applied        Scientific Catalog #AS-72092    -   15. EDTA: mix 14.12 g ethylenediaminetetraacetic acid (EDTA) to        approx. 70 ml dH₂O. Add 10

 N NaOH until EDTA dissolves. Adjust pH to 8.0. Adjust total volume to100 ml with dH₂O.

-   -   16. 1⁰ Antibody Dilution Buffer: mix 10 ml of 5% BSA in PBS        buffer with 89.5 ml TBST.    -   17. Anti-phosphotyrosine monoclonal antibody conjugated to        horseradish peroxidase (PY99

 HRP, Santa Cruz Biotech).

-   -   18. 2,2′-Azinobis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS,        Moss, Cat. No. ABST).    -   19. 10% SDS.        Procedure:    -   1. Coat Corning 96-well ELISA plates with 2 μg of polyEY peptide        in sterile PBS as described in

 step 3 of Materials and Reagents.

-   -   2. Remove unbound liquid from wells by inverting plate. Wash        once with TBST. Pat the plate on a paper towel to remove excess        liquid.    -   3. Add 100 μl of 1% BSA in PBS to each well. Incubate, with        shaking, for 1 hr. at room

 temperature.

-   -   4. Repeat step 2.    -   5. Soak wells with 50 mM HEPES (pH7.5) (150 μl/well).    -   6. Dilute test compound with dH₂O/4% DMSO to 4 times the desired        final assay concentration in

 96-well polypropylene plates.

-   -   7. Add 25 μl diluted test compound to ELISA plate. In control        wells, place 25 μl of dH₂O/4%

 DMSO.

-   -   8. Add 25 μl of 40 mM MnCl₂ with 4×ATP (2 μM) to each well.    -   9. Add 25 μl 0.5M EDTA to negative control wells.    -   10. Dilute GST-Flk1 to 0.005 μg (5 ng)/well with KDB.    -   11. Add 50 μl of diluted enzyme to each well.    -   12. Incubate, with shaking, for 15 minutes at room temperature.    -   13. Stop reaction by adding 50 μl of 250 mM EDTA (pH 8.0).    -   14. Wash 3× with TBST and pat plate on paper towel to remove        excess liquid.    -   15. Add 100 μl per well anti-phosphotyrosine HRP conjugate,        1:5,000 dilution in antibody dilution

 buffer. Incubate, with shaking, for 90 min. at room temperature.

-   -   16. Wash as in step 14.    -   17. Add 100 μl of room temperature ABTS solution to each well.    -   18. Incubate, with shaking, for 10 to 15 minutes. Remove any        bubbles.    -   19. Stop reaction by adding 20 μl of 10% SDS to each well.    -   20. Read results on Dynatech MR7000 ELISA reader with test        filter at 410 nM and reference filter at 630 nM.        pyk2 Bioassay

This assay is used to measure the in vitro kinase activity of HAepitope-tagged full length pyk2 (FL.pyk2-HA) in an ELISA assay.

Materials and Reagents:

-   -   1. Corning 96-well Elisa plates.    -   2. 12CA5 monoclonal anti-HA antibody (SUGEN, Inc.)    -   3. PBS (Dulbecco's Phosphate-Buffered Saline (Gibco Catalog        #450-1300EB)    -   4. TBST Buffer: for 1 L, mix 8.766 g NaCl, 6.057 g TRIS and 1 ml        of 0.1% Triton X-100 in

 approx. 900 ml dH₂O. Adjust pH to 7.2, bring volume to 1 L.

-   -   5. Blocking Buffer: for 1 L, mix 100 g 10% BSA, 12.1 g 100 mM        TRIS, 58.44 g 1M NaCl and 10

 mL of 1% TWEEN-20.

-   -   6. FL.pyk2-HA from sf9 cell lysates (SUGEN, Inc.).    -   7. 4% DMSO in MilliQue H₂O.    -   8. 10 mM ATP in dH₂O.    -   9. 1M MnCl₂.    -   10. 1M MgCl₂.    -   11. 1M Dithiothreitol (DTT).    -   12. 10× Kinase buffer phosphorylation: mix 5.0 ml 1M Hepes (pH        7.5), 0.2 ml 1M    -    MnCl₂, 1.0 ml 1    -    M MgCl₂, 1.0 ml 10% Triton X-100 in 2.8 ml dH₂O. Just prior to        use, add 0.1 ml 1M DTT.    -   13. NUNC 96-well V bottom polypropylene plates.    -   14. 500 mM EDTA in dH₂O.    -   15. Antibody dilution buffer: for 100 mL, 1 mL 5% BSA/PBS and 1        mL 10% Tween-20 in 88 mL    -    TBS.    -   16. HRP-conjugated anti-Ptyr PY99), Santa Cruz Biotech Cat. No.        SC-7020.    -   17. ABTS, Moss, Cat. No. ABST-2000.    -   18. 10% SDS.        Procedure:    -   1. Coat Corning 96 well ELISA plates with 0.5 μg per well 12CA5        anti-HA antibody in 100 μl

 PBS. Store overnight at 4° C.

-   -   2. Remove unbound HA antibody from wells by inverting plate.        Wash plate with dH₂O. Pat the

 plate on a paper towel to remove excess liquid.

-   -   3. Add 150 μl Blocking Buffer to each well. Incubate, with        shaking, for 30 min at room

 temperature.

-   -   4. Wash plate 4× with TBS-T.    -   5. Dilute lysate in PBS (1.5 μg lysate/100 μl PBS).    -   6. Add 100 μl of diluted lysate to each well. Shake at room        temperature for 1 hr.    -   7. Wash as in step 4.    -   8. Add 50 μl of 2× kinase Buffer to ELISA plate containing        captured pyk2-HA.    -   9. Add 25 μL of 400 μM test compound in 4% DMSO to each well.        For control wells use 4%

 DMSO alone.

-   -   10. Add 25 μL of 0.5 M EDTA to negative control wells.    -   11. Add 25 μl of 20 μM ATP to all wells. Incubate, with shaking,        for 10 minutes.    -   12. Stop reaction by adding 25 μl 500 mM EDTA (pH 8.0) to all        wells.    -   13. Wash as in step 4.    -   14. Add 100 μL HRP conjugated anti-Ptyr diluted 1:6000 in        Antibody Dilution Buffer to each well.

 Incubate, with shaking, for 1 hr. at room temperature.

-   -   15. Wash plate 3× with TBST and 1× with PBS.    -   16. Add 100 μL of ABST solution to each well.    -   17. If necessary, stop the development reaction by adding 20 μL        10% SDS to each well.    -   18. Read plate on ELISA reader with test filter at 410 nM and        reference filter at 630 nM.        FGFR1 Bioassay

This assay is used to measure the in vitro kinase activity of FGF1-R inan ELISA assay.

Materials and Reagents:

-   -   1. Costar 96-well Elisa plates (Corning Catalog #3369).    -   2. Poly(Glu-Tyr) (Sigma Catalog #PO275).    -   3. PBS (Gibco Catalog #450-1300EB)    -   4. 50 mM Hepes Buffer Solution.    -   5. Blocking Buffer (5% BSA/PBS).    -   6. Purified GST-FGFR1 (SUGEN, Inc.)    -   7. Kinase Dilution Buffer.

 Mix 500 μl 1M Hepes (GIBCO), 20 μl 5% BSA/PBS, 10 μl 100 mM sodiumorthovanadate and

-   -    50 μl 5M NaCl.    -   8. 10 mM ATP    -   9. ATP/MnCl₂ phosphorylation mix: mix 20 μL ATP, 400 μL 1 M        MnCl₂ and 9.56 ml dH₂O.    -   10. NUNC 96-well V bottom polypropylene plates (Applied        Scientific Catalog #AS-72092).    -   11. 0.5M EDTA.    -   12. 0.05% TBST    -    Add 500 μL TWEEN to 1 liter TBS.    -   13. Rabbit polyclonal anti-phosphotyrosine serum (SUGEN, Inc.).    -   14. Goat anti-rabbit IgG peroxidase conjugate (Biosource,        Catalog #ALI0404).    -   15. ABTS Solution.    -   16. ABTS/H₂O₂ solution.        Procedure:    -   1. Coat Costar 96 well ELISA plates with 1 μg per well        Poly(Glu,Tyr) in 100 μl PBS. Store    -    overnight at 4° C.    -   2. Wash coated plates once with PBS.    -   3. Add 150 μL of 5% BSA/PBS Blocking Buffer to each well.        Incubate, with shaking, for 1    -    hr. room temperature.    -   4. Wash plate 2× with PBS, then once with 50 mM Hepes. Pat        plates on a paper towel to remove    -    excess liquid and bubbles.    -   5. Add 25 μL of 0.4 mM test compound in 4% DMSO or 4% DMSO alone        (controls) to plate.    -   6. Dilute purified GST-FGFR1 in Kinase Dilution Buffer (5 ng        kinase/50 ul KDB/well).    -   7. Add 50 μL of diluted kinase to each well.    -   8. Start kinase reaction by adding 25 μl/well of freshly        prepared ATP/Mn++(0.4 ml 1 M MnCl₂, 40 μL 10 mM ATP, 9.56 ml        dH₂O), freshly prepared).    -   9. This is a fast kinase reaction and must be stopped with 25 μL        of 0.5M EDTA in a manner    -    similar to the addition of ATP.    -   10. Wash plate 4× with fresh TBST.    -   11. Make up Antibody Dilution Buffer: Per 50 ml:    -   Mix 5 ml of 5% BSA, 250 μl of 5% milk and 50 μl of 100 mM sodium        vanadate, bring to final    -    volume with 0.05% TBST.    -   12. Add 100 μl per well of anti-phosphotyrosine (1:10000        dilution in ADB). Incubate, with shaking    -    for 1 hr. at room temperature.    -   13. Wash as in step 10.    -   14. Add 100 μl per well of Biosource Goat anti-rabbit IgG        peroxidase conjugate (1:6000 dilution in    -    ADB). Incubate, with shaking for 1 hr. at room temperature.    -   15. Wash as in step 10 and then with PBS to remove bubbles and        excess TWEEN.    -   16. Add 100 μl of ABTS/H₂O₂ solution to each well.    -   17. Incubate, with shaking, for 10 to 20 minutes. Remove any        bubbles.    -   18. Read assay on Dynatech MR7000 elisa reader: test filter at        410 nM, reference filtrate 630 nM.        EGFR Bioassay

This assay is used to the in vitro kinase activity of FGF1-R in an ELISAassay.

Materials and Reagents:

-   -   1. Corning 96-well Elisa plates.    -   2. SUMO1 monoclonal anti-EGFR antibody (SUGEN, Inc.).    -   3. PBS    -   4. TBST Buffer    -   5. Blocking Buffer: for 100 ml, mix 5.0 g Carnation Instant        Non-fat Milk® with 100 ml of PBS.    -   6. A431 cell lysate (SUGEN, Inc.).    -   7. TBS Buffer:    -   8. TBS+10% DMSO: for 1 L, mix 1.514 g TRIS, 2.192 g NaCl and 25        ml DMSO; bring to 1 liter    -    total volume with dH₂O.    -   9. ATP (Adenosine-5′-triphosphate, from Equine muscle, Sigma        Cat. No. A-5394), 1.0 mM    -    solution in dH₂O. This reagent should be made up immediately        prior to use and kept on ice.    -   10. 1.0 mM MnCl₂.    -   11. ATP/MnCl₂ phosphorylation mix: to make 10 ml, mix 300 μl of        1 mM ATP, 500 μl MnCl₂ and    -    9.2 ml dH₂O. Prepare just prior to use, keep on ice.    -   12. NUNC 96-well V bottom polypropylene plates.    -   13. EDTA.    -   14. Rabbit polyclonal anti-phosphotyrosine serum (SUGEN, Inc.).    -   15. Goat anti-rabbit IgG peroxidase conjugate (Biosource Cat.        No. ALI0404).    -   16. ABTS.    -   17. 30% Hydrogen peroxide.    -   18. ABTS/H₂O₂.    -   19. 0.2 M HCl.        Procedure:    -   1. Coat Corning 96 well ELISA plates with 0.5 μg SUMO1 in 100 μl        PBS per well, store    -    overnight at 4° C.    -   2. Remove unbound SUMO1 from wells by inverting plate to remove        liquid. Wash 1× with dH₂O.    -    Pat the plate on a paper towel to remove excess liquid.    -   3. Add 150 μl of Blocking Buffer to each well. Incubate, with        shaking, for 30 min. at room    -    temperature.    -   4. Wash plate 3× with deionized water, then once with TBST. Pat        plate on a paper towel to    -    remove excess liquid and bubbles.    -   5. Dilute lysate in PBS (7 μg lysate/100 μl PBS).    -   6. Add 100 μl of diluted lysate to each well. Shake at room        temperature for 1 hr.    -   7. Wash plates as in 4, above.    -   8. Add 120 μl TBS to ELISA plate containing captured EGFR.    -   9. Dilute test compound 1:10 in TBS, place in well    -   10. Add 13.5 μl diluted test compound to ELISA plate. To control        wells, add 13.5 μl TBS in 10%    -    DMSO.    -   11. Incubate, with shaking, for 30 minutes at room temperature.    -   12. Add 15 μl phosphorylation mix to all wells except negative        control well. Final well volume    -    should be approximately 150 μl with 3 μM ATP/5 mM MnCl₂ final        concentration in each well.    -    Incubate with shaking for 5 minutes.    -   13. Stop reaction by adding 16.5 μl of EDTA solution while        shaking. Shake for additional 1 min.    -   14. Wash 4× with deionized water, 2× with TBST.    -   15. Add 100 μl anti-phosphotyrosine (1:3000 dilution in TBST)        per well. Incubate, with shaking,    -    for 30-45 min. at room temperature.    -   16. Wash as in 4, above.    -   17. Add 100 μl Biosource Goat anti-rabbit IgG peroxidase        conjugate (1:2000 dilution in TBST) to    -    each well. Incubate with shaking for 30 min. at room        temperature.    -   18. Wash as in 4, above.    -   19. Add 100 μl of ABTS/H₂O₂ solution to each well.    -   20. Incubate 5 to 10 minutes with shaking. Remove any bubbles.    -   21. If necessary, stop reaction by adding 100 μl 0.2 M HCl per        well.    -   22. Read assay on Dynatech MR7000 ELISA reader: test filter at        410 nM, reference filter at 630 nM.        PDGFR Bioassay

This assay is used to the in vitro kinase activity of FGF1-R in an ELISAassay.

Materials and Reagents:

-   -   1. Corning 96-well Elisa plates    -   2. 28D4C10 monoclonal anti-PDGFR antibody (SUGEN, Inc.).    -   3. PBS.    -   4. TBST Buffer.    -   5. Blocking Buffer (same as for EGFR bioassay).    -   6. PDGFR-β expressing NIH 3T3 cell lysate (SUGEN, Inc.).    -   7. TBS Buffer.    -   8. TBS+10% DMSO.    -   9. ATP.    -   10. MnCl₂.    -   11. Kinase buffer phosphorylation mix: for 10 ml, mix 250 μl 1M        TRIS, 200 μl 5M    -    NaCl, 100 μl 1M    -    MnCl₂ and 50 μl 100 mM Triton X-100 in enough dH₂O to make 10        ml.    -   12. NUNC 96-well V bottom polypropylene plates.    -   13. EDTA.    -   14. Rabbit polyclonal anti-phosphotyrosine serum (SUGEN, Inc.).    -   15. Goat anti-rabbit IgG peroxidase conjugate (Biosource Cat.        No. ALI0404).    -   16. ABTS.    -   17. Hydrogen peroxide, 30% solution.    -   18. ABTS/H₂O₂.    -   19. 0.2 M HCl.        Procedure:    -   1. Coat Corning 96 well ELISA plates with 0.5 μg 28D4C10 in 100        μl PBS per    -    well, store    -    overnight at 4° C.    -   2. Remove unbound 28D4C10 from wells by inverting plate to        remove liquid. Wash 1× with dH₂O. Pat the plate on a paper towel        to remove excess liquid.    -   3. Add 150 μl of Blocking Buffer to each well. Incubate for 30        min. at room temperature with        -   shaking.    -   4. Wash plate 3× with deionized water, then once with TBST. Pat        plate on a paper towel to    -    remove excess liquid and bubbles.    -   5. Dilute lysate in HNTG (10 μg lysate/100 μl HNTG).    -   6. Add 100 μl of diluted lysate to each well. Shake at room        temperature for 60 min.    -   7. Wash plates as described in Step 4.    -   8. Add 80 μl working kinase buffer mix to ELISA plate containing        captured PDGFR.    -   9. Dilute test compound 1:10 in TBS in 96-well polypropylene        plates.    -   10. Add 10 μl diluted test compound to ELISA plate. To control        wells, add 10 μl TBS+10%    -    DMSO. Incubate with shaking for 30 minutes at room temperature.    -   11. Add 10 μl ATP directly to all wells except negative control        well (final well volume should be    -    approximately 100 μl with 20 μM ATP in each well.) Incubate 30        minutes with shaking.    -   12. Stop reaction by adding 10 μl of EDTA solution to each well.    -   13. Wash 4× with deionized water, twice with TBST.    -   14. Add 100 μl anti-phosphotyrosine (1:3000 dilution in TBST)        per well. Incubate with shaking for    -    30-45 min. at room temperature.    -   15. Wash as in Step 4.    -   16. Add 100 μl Biosource Goat anti-rabbit IgG peroxidase        conjugate (1:2000 dilution in TBST) to    -    each well. Incubate with shaking for 30 min. at room        temperature.    -   17. Wash as in Step 4.    -   18. Add 100 μl of ABTS/H₂O₂ solution to each well.    -   19. Incubate 10 to 30 minutes with shaking. Remove any bubbles.    -   20. If necessary stop reaction with the addition of 100 μl 0.2 M        HCl per well.    -   21. Read assay on Dynatech MR7000 ELISA reader with test filter        at 410 nM and reference filter    -    at 630 nM.        Cellular HER-2 Kinase Assay

This assay is used to measure HER-2 kinase activity in whole cells in anELISA format.

Materials and Reagents:

-   -   1. DMEM (GIBCO Catalog #11965-092).    -   2. Fetal Bovine Serum (FBS, GIBCO Catalog #16000-044), heat        inactivated in a water bath for    -    30 min. at 56° C.    -   3. Trypsin (GIBCO Catalog #25200-056).    -   4. L-Glutamine (GIBCO Catalog #25030-081)    -   5. HEPES (GIBCO Catalog #15630-080).    -   6. Growth Media    -   Mix 500 ml DMEM, 55 ml heat inactivated FBS, 10 ml HEPES and 5.5        ml L-Glutamine.    -   7. Starve Media    -   Mix 500 ml DMEM, 2.5 ml heat inactivated FBS, 10 ml HEPES and        5.5 ml L-Glutamine.    -   8. PBS.    -   9. Flat Bottom 96-well Tissue Culture Micro Titer Plates        (Corning Catalog #25860).    -   10. 15 cm Tissue Culture Dishes (Corning Catalog #08757148).    -   11. Corning 96-well ELISA Plates.    -   12. NUNC 96-well V bottom polypropylene plates.    -   13. Costar Transfer Cartridges for the Transtar 96 (Costar        Catalog #7610).    -   14. SUMO 1: monoclonal anti-EGFR antibody (SUGEN, Inc.).    -   15. TBST Buffer.    -   16. Blocking Buffer: 5% Carnation Instant Milk® in PBS.    -   17. EGF Ligand: EGF-201, Shinko American, Japan. Suspend powder        in 100 uL of 10 mM HCl.    -    Add 100 uL 10 mM NaOH. Add 800 uL PBS and transfer to an        Eppendorf tube, store at −20° C.    -    until ready to use.    -   18. HNTG Lysis Buffer    -    For Stock 5× HNTG, mix 23.83 g Hepes, 43.83 g NaCl, 500 ml        glycerol and 100 ml Triton X-    -    100 and enough dH₂O to make 1 L of total solution.    -    For 1× HNTG*, mix 2 ml HNTG, 100 μL 0.1M Na₃VO₄, 250 μL 0.2M        Na₄P₂O₇ and 100 μL    -    EDTA.    -   19. EDTA.    -   20. Na₃VO₄. To make stock solution, mix 1.84 g Na₃VO₄ with 90 ml        dH    -    2O. Adjust pH to 10. Boil    -    in microwave for one minute (solution becomes clear). Cool to        room temperature. Adjust pH    -    to 10. Repeat heating/cooling cycle until pH remains at 10.    -   21. 200 mM Na₄P₂O₇.    -   22. Rabbit polyclonal antiserum specific for phosphotyrosine        (anti-Ptyr antibody, SUGEN, Inc.).    -   23. Affinity purified antiserum, goat anti-rabbit IgG antibody,        peroxidase conjugate (Biosource Cat # ALI0404).    -   24. ABTS Solution.    -   25. 30% Hydrogen peroxide solution.    -   26. ABTS/H₂O₂.    -   27. 0.2 M HCl.        Procedure:    -   1. Coat Corning 96 well ELISA plates with SUMO1 at 1.0 ug per        well in PBS, 100 ul final    -    volume/well. Store overnight at 4° C.    -   2. On day of use, remove coating buffer and wash plate 3 times        with dH₂O and once with TBST    -    buffer. All washes in this assay should be done in this manner,        unless otherwise specified.    -   3. Add 100 ul of Blocking Buffer to each well. Incubate plate,        with shaking, for 30 min. at room    -    temperature. Just prior to use, wash plate.    -   4. Use EGFr/HER-2 chimera/3T3-C7 cell line for this assay.    -   5. Choose dishes having 80-90% confluence. Collect cells by        trypsinization and centrifuge at 1000 rpm at room temperature        for 5 min.    -   6. Resuspend cells in starve medium and count with trypan blue.        Viability above 90% is    -    required. Seed cells in starve medium at a density of 2,500        cells per well, 90 ul per well, in a 96 well microtiter plate.        Incubate seeded cells overnight at 37° under 5% CO₂.    -   7. Start the assay two days after seeding.    -   8. Test compounds are dissolved in 4% DMSO. Samples are then        further diluted directly on    -    plates with starve-DMEM. Typically, this dilution will be 1:10        or greater. All wells are then    -    transferred to the cell plate at a further 1:10 dilution (10 μl        sample and media into 90 μl of    -    starve media. The final DMSO concentration should be 1% or        lower. A standard serial    -    dilution may also be used.    -   9. Incubate under 5% CO₂ at 37° C. for 2 hours.    -   10. Prepare EGF ligand by diluting stock EGF (16.5 uM) in warm        DMEM to 150 nM.    -   11. Prepare fresh HNTG* sufficient for 100 ul per well; place on        ice.    -   12. After 2 hour incubation with test compound, add prepared EGF        ligand to cells, 50 ul per well,    -    for a final concentration of 50 nM. Positive control wells        receive the same amount of EGF.    -    Negative controls do not receive EGF. Incubate at 37° C. for 10        min.    -   13. Remove test compound, EGF, and DMEM. Wash cells once with        PBS.    -   14. Transfer HNTG* to cells, 100 ul per well. Place on ice for 5        minutes. Meanwhile, remove    -    blocking buffer from ELISA plate and wash.    -   15. Scrape cells from plate with a micropipettor and homogenize        cell material by repeatedly    -    aspirating and dispensing the HNTG* lysis buffer. Transfer        lysate to a coated, blocked,    -    washed ELISA plate. Or, use a Costar transfer cartridge to        transfer lysate to the plate.    -   16. Incubate, with shaking, at room temperature for 1 hr.    -   17. Remove lysate, wash. Transfer freshly diluted anti-Ptyr        antibody (1:3000 in TBST) to ELISA    -    plate, 100 ul per well.    -   18. Incubate, with shaking, at room temperature, for 30 min.    -   19. Remove anti-Ptyr antibody, wash. Transfer freshly diluted        BIOSOURCE antibody to ELISA    -    plate (1:8000 in TBST, 100 ul per well).    -   20. Incubate, with shaking, at room temperature for 30 min.    -   21. Remove BIOSOURCE antibody, wash. Transfer freshly prepared        ABTS/H₂O₂ solution to    -    ELISA plate, 100 ul per well.    -   22. Incubate, with shaking, for 5-10 minutes. Remove any        bubbles.    -   23. Stop reaction with the addition of 100 ul of 0.2M HCl per        well.    -   24. Read assay on Dynatech MR7000 ELISA reader with test filter        set at 410 nM and reference    -    filter at 630 nM.        cdk2/cyclin A Assay

This assay is used to measure the in vitro serine/threonine kinaseactivity of human cdk2/cyclin A in a Scintillation Proximity Assay(SPA).

Materials and Reagents.

-   -   1. Wallac 96-well polyethylene terephthalate (flexi) plates        (Wallac Catalog #1450-401).    -   2. Amersham Redivue [γ³³P] ATP (Amersham catalog #AH 9968).    -   3. Amersham streptavidin coated polyvinyltoluene SPA beads        (Amersham catalog    -    #RPNQ0007). The beads should be reconstituted in PBS without        magnesium or calcium, at    -    20 mg/ml.    -   4. Activated cdk2/cyclin A enzyme complex purified from Sf9        cells (SUGEN, Inc.).    -   5. Biotinylated peptide substrate (Debtide). Peptide        biotin-X-PKTPKKAKKL is dissolved in dH₂O    -    at a concentration of 5 mg/ml.    -   6. Peptide/ATP Mixture: for 10 ml, mix 9.979 ml dH₂O, 0.00125 ml        “cold” ATP, 0.010 ml Debtide    -    and 0.010 ml γ³³P ATP. The ultimate concentration per well will        be 0.5 μM “cold” ATP, 0.1 μg Debtide and 0.2 μCi γ³³P ATP.    -   7. Kinase buffer: for 10 ml, mix 8.85 ml dH₂O, 0.625 ml TRIS (pH        7.4), 0.25 ml 1 M MgCl₂, 0.25    -    ml 10% NP40 and 0.025 ml 1 M DTT, added fresh just prior to        use.    -   8. 10 mM ATP in dH₂O.    -   9. 1 M Tris, pH adjusted to 7.4 with HCl.    -   10. 1M MgCl₂.    -   11. 1 M DTT.    -   12. PBS (Gibco Catalog #14190-144).    -   13. 0.5M EDTA.    -        -   14. Stop solution: For 10 ml, mix 9.25 ml PBS, 0.005 ml 100 mM        ATP, 0.1 ml 0.5 M EDTA, 0.1    -    ml 10% Triton X-100 and 1.25 ml of 20 mg/ml SPA beads.        Procedure:    -   1. Prepare solutions of test compounds at 5× the desired final        concentration in 5% DMSO. Add 10 ul to each well. For negative        controls, use 10 ul 5% DMSO alone in wells.    -   2. Dilute 5 μl of cdk2/cyclin A solution with 2.1 ml 2× kinase        buffer.    -   3. Add 20 ul enzyme to each well.    -   4. Add 10 μL of 0.5 M EDTA to the negative control wells.    -   5. To start kinase reaction, add 20 μL of peptide/ATP mixture to        each well. Incubate for 1 hr.    -    without shaking.    -   6. Add 200 μl stop solution to each well.    -   7. Hold at least 10 min.    -   8. Spin plate at approx. 2300 rpm for 3-5 min.    -   9. Count plate using Trilux or similar reader.        Met Transphosphorylation Assay

This assay is used to measure phosphotyrosine levels on a poly(glutamicacid:tyrosine (4:1)) substrate as a means for identifyingagonists/antagonists of met transphosphorylation of the substrate.

Materials and Reagents:

-   -   1. Corning 96-well Elisa plates, Corning Catalog #25805-96.    -   2. Poly(glu, tyr) 4:1, Sigma, Cat. No; P 0275.    -   3. PBS, Gibco Catalog #450-1300EB    -   4. 50 mM HEPES    -   5. Blocking Buffer: Dissolve 25 g Bovine Serum Albumin, Sigma        Cat. No A-7888, in 500 ml    -    PBS, filter through a 4 μm filter.    -   6. Purified GST fusion protein containing the Met kinase domain,        Sugen, Inc.    -   7. TBST Buffer.    -   8. 10% aqueous (MilliQue H₂O) DMSO.    -   9. 10 mM aqueous (dH₂O) Adenosine-5′-triphosphate, Sigma Cat.        No. A-5394.    -   10. 2× Kinase Dilution Buffer: for 100 ml, mix 10 mL 1M HEPES at        pH 7.5 with 0.4 mL 5%    -    BSA/PBS, 0.2 mL 0.1 M sodium orthovanadate and 1 mL 5M sodium        chloride in 88.4 mL    -    dH₂O.    -   11. 4× ATP Reaction Mixture: for 10 mL, mix 0.4 mL 1 M manganese        chloride and 0.02 mL 0.1    -    M ATP in 9.56 mL dH₂O.    -        -   12. 4× Negative Controls Mixture: for 10 mL, mix 0.4 mL 1 M        manganese chloride in 9.6 mL    -    dH₂O.    -   13. NUNC 96-well V bottom polypropylene plates, Applied        Scientific Catalog #S-72092    -   14. 500 mM EDTA.    -   15. Antibody Dilution Buffer: for 100 mL, mix 10 mL 5% BSA/PBS,        0.5 mL 5% Carnation Instant    -    Milk® in PBS and 0.1 mL 0.1 M sodium orthovanadate in 88.4 mL        TBST.    -   16. Rabbit polyclonal antophosphotyrosine antibody, Sugen, Inc.    -   17. Goat anti-rabbit horseradish peroxidase conjugated antibody,        Biosource, Inc.    -   18. ABTS Solution: for 1 L, mix 19.21 g citric acid, 35.49 g        Na₂HPO₄ and 500 mg    -    ABTS with sufficient dH₂O to make 1 L.    -   19. ABTS/H₂O₂: mix 15 mL ABST solution with 2 μL H₂O₂ five        minutes    -    before use.    -   20. 0.2 M HCl        Procedure:    -   1. Coat ELISA plates with 2 μg Poly(Glu-Tyr) in 100 μL PBS,        store overnight at 4° C.    -   2. Block plate with 150 μL of 5% BSA/PBS for 60 min.    -   3. Wash plate twice with PBS, once with 50 mM Hepes buffer pH        7.4.    -   4. Add 50 μl of the diluted kinase to all wells. (Purified        kinase is diluted with Kinase Dilution    -    Buffer. Final concentration should be 10 ng/well.)    -   5. Add 25 μL of the test compound (in 4% DMSO) or DMSO alone (4%        in dH₂O) for    -    controls to plate.    -   6. Incubate the kinase/compound mixture for 15 minutes.    -   7. Add 25 μL of 40 mM MnCl₂ to the negative control wells.    -   8. Add 25 μL ATP/MnCl₂ mixture to the all other wells (except        the negative controls). Incubate for 5 min.    -   9. Add 25 μL 500 mM EDTA to stop reaction.    -   10. Wash plate 3× with TBST.    -   11. Add 100 μL rabbit polyclonal anti-Ptyr diluted 1:10,000 in        Antibody Dilution Buffer to each    -    well. Incubate, with shaking, at room temperature for one hour.    -   12. Wash plate 3× with TBST.    -   13. Dilute Biosource HRP conjugated anti-rabbit antibody 1:6,000        in Antibody Dilution buffer.    -    Add 100 μL per well and incubate at room temperature, with        shaking, for one hour.    -   14. Wash plate 1× with PBS.    -   15. Add 100 μl of ABTS/H₂O₂ solution to each well.    -   16. If necessary, stop the development reaction with the        addition of 100 μl of 0.2M HCl per well.    -   17. Read plate on Dynatech MR7000 elisa reader with the test        filter at 410 nM and the reference    -    filter at 630 nM.        IGF-1 Transphosphorylation Assay        This assay is used to measure the phosphotyrosine level in        poly(glutamic acid:tyrosine)(4:1) for the identification of        agonists/antagonists of gst-IGF-1 transphosphorylation of a        substrate.        Materials and Reagents:    -   1. Corning 96-well Elisa plates.    -   2. Poly (Glu-tyr) (4:1), Sigma Cat. No. P 0275.    -   3. PBS, Gibco Catalog #450-1300EB.    -   4. 50 mM HEPES    -   5. TBB Blocking Buffer: for 1 L, mix 100 g BSA, 12.1 gTRIS (pH        7.5), 58.44 g sodium chloride    -    and 10 mL 1% TWEEN-20.    -   6. Purified GST fusion protein containing the IGF-1 kinase        domain (Sugen, Inc.)    -   7. TBST Buffer: for 1 L, mix 6.057 g Tris, 8.766 g sodium        chloride and 0.5 ml TWEEN-20 with    -    enough dH₂O to make 1 liter.    -   8. 4% DMSO in Milli-Q H₂O.    -   9. 10 mM ATP in dH₂O.    -   10. 2×Kinase Dilution Buffer: for 100 mL, mix 10 mL 1 M HEPES        (pH 7.5), 0.4 mL 5% BSA in    -    dH₂O, 0.2 mL 0.1 M sodium orthovanadate and 1 mL 5 M sodium        chloride with enough dH₂O    -    to make 100 mL.    -   11. 4×ATP Reaction Mixture: for 10 mL, mix 0.4 mL 1 M MnCl₂ and        0.008 mL 0.01 M ATP and 9.56 mL dH₂O.    -   12. 4× Negative Controls Mixture: mix 0.4 mL 1 M manganese        chloride in 9.60 mL dH₂O.    -   13. NUNC 96-well V bottom polypropylene plates.    -   14. 500 mM EDTA in dH₂O.    -   15. Antibody Dilution Buffer: for 100 mL, mix 10 mL 5% BSA in        PBS, 0.5 mL 5% Carnation    -    Instant Non-fat Milk® in PBS and 0.1 mL 0.1 M sodium        orthovanadate in 88.4 mL TBST.    -   16. Rabbit Polyclonal antiphosphotyrosine antibody, Sugen, Inc.    -   17. Goat anti-rabbit HRP conjugated antibody, Biosource.    -   18. ABTS Solution.    -   20. ABTS/H₂O₂: mix 15 mL ABTS with 2 μL H₂O₂ 5 minutes before        using.    -   21. 0.2 M HCl in dH₂O.        Procedure:    -   1. Coat ELISA plate with 2.0 μg/well Poly(Glu, Tyr) 4:1 (Sigma        P0275) in 100 μl PBS. Store    -    plate overnight at 4° C.    -   2. ash plate once with PBS.    -   3. Add 100 μl of TBB Blocking Buffer to each well. Incubate        plate for 1 hour with shaking at    -    room temperature.    -   4. Wash plate once with PBS, then twice with 50 mM Hepes buffer        pH 7.5.    -   5. Add 25 μL of test compound in 4% DMSO (obtained by diluting a        stock solution of 10    -    mM test compound in 100% DMSO with dH₂O) to plate.    -   6. Add 10.0 ng of gst-IGF-1 kinase in 50 μl Kinase Dilution        Buffer) to all wells.    -   7. Start kinase reaction by adding 25 μl 4× ATP Reaction Mixture        to all test wells and positive    -    control wells. Add 25 μl 4× Negative Controls Mixture to all        negative control wells. Incubates    -    for 10 minutes with shaking at room temperature.    -   8. Add 25 μl 0.5M EDTA (pH 8.0) to all wells.    -   9. Wash plate 4× with TBST Buffer.    -   10. Add rabbit polyclonal anti-phosphotyrosine antisera at a        dilution of 1:10,000 in 100 μl Antibody    -    Dilution Buffer to all wells. Incubate, with shaking, at room        temperature for 1 hour.    -   11. Wash plate as in step 9.    -   12. Add 100 μL Biosource anti-rabbit HRP at a dilution of        1:10,000 in Antibody dilution buffer to    -    all wells. Incubate, with shaking, at room temperature for 1        hour.    -   13. Wash plate as in step 9, follow with one wash with PBS to        reduce bubbles and excess    -    Tween-20.    -   14. Develop by adding 100 μl/well ABTS/H₂O₂ to each well.    -   15. After about 5 minutes, read on ELISA reader with test filter        at 410 nm and referenced filter at 630 nm.        BrdU Incorporation Assays

The following assays use cells engineered to express a selected receptorand then evaluate the effect of a compound of interest on the activityof ligand-induced DNA synthesis by determining BrdU incorporation intothe DNA.

The following materials, reagents and procedure are general to each ofthe following BrdU incorporation assays. Variances in specific assaysare noted.

Materials and Reagents:

-   -   1. The appropriate ligand.    -   2. The appropriate engineered cells.    -   3. BrdU Labeling Reagent: 10 mM, in PBS (pH 7.4)(Boehringer        Mannheim, Germany).    -   4. FixDenat: fixation solution (ready to use)(Boehringer        Mannheim, Germany).    -   5. Anti-BrdU-POD: mouse monoclonal antibody conjugated with        peroxidase (Boehringer    -    Mannheim, Germany).    -   6. TMB Substrate Solution: tetramethylbenzidine (TMB, Boehringer        Mannheim, Germany).    -   7. PBS Washing Solution: 1× PBS, pH 7.4.    -   8. Albumin, Bovine (BSA), fraction V powder (Sigma Chemical Co.,        USA).        General Procedure:    -   1. Cells are seeded at 8000 cells/well in 10% CS, 2 mM Gln in        DMEM, in a 96 well plate. Cells are    -    incubated overnight at 37° C. in 5% CO₂.    -   2. After 24 hours, the cells are washed with PBS, and then are        serum-starved in serum free medium    -    (0% CS DMEM with 0.1% BSA) for 24 hours.    -   3. On day 3, the appropriate ligand and the test compound are        added to the cells    -    simultaneously. The negative control wells receive serum free        DMEM with 0.1% BSA only;    -    the positive control cells receive the ligand but no test        compound. Test compounds are    -    prepared in serum free DMEM with ligand in a 96 well plate, and        serially diluted for 7 test    -    concentrations.    -   4. After 18 hours of ligand activation, diluted BrdU labeling        reagent (1:100 in DMEM, 0.1%    -    BSA) is added and the cells are incubated with BrdU (final        concentration=10 μM) for 1.5    -    hours.    -   5. After incubation with labeling reagent, the medium is removed        by decanting and tapping the    -    inverted plate on a paper towel. FixDenat solution is added (50        μl/well) and the plates are    -    incubated at room temperature for 45 minutes on a plate shaker.    -   6. The FixDenat solution is thoroughly removed by decanting and        tapping the inverted plate on    -    a paper towel. Milk is added (5% dehydrated milk in PBS, 200        μl/well) as a blocking solution    -    and the plate is incubated for 30 minutes at room temperature        on a plate shaker.    -   7. The blocking solution is removed by decanting and the wells        are washed once with PBS.    -    Anti-BrdU-POD solution (1:200 dilution in PBS, 1% BSA) is added        (50 μl/well) and the plate    -    is incubated for 90 minutes at room temperature on a plate        shaker.    -   8. The antibody conjugate is thoroughly removed by decanting and        rinsing the wells 5 times with    -    PBS, and the plate is dried by inverting and tapping on a paper        towel.    -   9. TMB substrate solution is added (100 μl/well) and incubated        for 20 minutes at room    -    temperature on a plate shaker until color development is        sufficient for photometric detection.

10. The absorbance of the samples are measured at 410 nm (in “dualwavelength” mode with a

-   -    filter reading at 490 nm, as a reference wavelength) on a        Dynatech ELISA plate reader.        EGF-Induced BrdU Incorporation Assay        Materials and Reagents:    -   1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).    -   2. 3T3/EGFRc7.        EGF-Induced Her-2-Driven BrdU Incorporation Assay        Materials and Reagents:    -   1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).    -   2. 3T3/EGFr/Her2/EGFr (EGFr with a Her-2 kinase domain).        EGF-Induced Her-4-driven BrdU Incorporation Assay        Materials and Reagents:    -   1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).    -   2. 3T3/EGFr/Her4/EGFr (EGFr with a Her-4 kinase domain).        PDGF-Induced BrdU Incorporation Assay        Materials and Reagents:    -   1. Human PDGF B/B (Boehringer Mannheim, Germany).    -   2. 3T3/EGFRc7.        FGF-Induced BrdU Incorporation Assay        Materials and Reagents:    -   1. Human FGF2/bFGF (Gibco BRL, USA).    -   2. 3T3c7/EGFr        IGF1-Induced BrdU Incorporation Assay        Materials and Reagents:    -   1. Human, recombinant (G511, Promega Corp., USA)    -   2. 3T3/IGF1r.        Insulin-Induced BrdU Incorporation Assay        Materials and Reagents:    -   1. Insulin, crystalline, bovine, Zinc (13007, Gibco BRL, USA).    -   2. 3T3/H25.        HGF-Induced BrdU Incorporation Assay

Materials and Reagents:

-   -   1. Recombinant human HGF (Cat. No. 249-HG, R&D Systems, Inc.        USA).    -   2. BxPC-3 cells (ATCC CRL-1687).        Procedure:    -   1. Cells are seeded at 9000 cells/well in RPMI 10% FBS in a 96        well plate. Cells are incubated    -    overnight at 37° C. in 5% CO₂.    -   2. After 24 hours, the cells are washed with PBS, and then are        serum starved in 100 μl serum-free    -    medium (RPMI with 0.1% BSA) for 24 hours.    -   3. On day 3, 25 μl containing ligand (prepared at 1 μg/ml in        RPMI with 0.1% BSA; final HGF    -    conc. is 200 ng/ml) and test compounds are added to the cells.        The negative control wells    -    receive 25 μl serum-free RPMI with 0.1% BSA only; the positive        control cells receive the    -    ligand (HGF) but no test compound. Test compounds are prepared        at 5 times their final    -    concentration in serum-free RPMI with ligand in a 96 well        plate, and serially diluted to give 7    -    test concentrations. Typically, the highest final concentration        of test compound is 100 μM,    -    and 1:3 dilutions are used (i.e. final test compound        concentration range is 0.137-100 μM).    -   4. After 18 hours of ligand activation, 12.5 μl of diluted BrdU        labeling reagent (1:100 in RPMI, 0.1% BSA) is added to each well        and the cells are incubated with BrdU (final concentration is 10        μM) for 1 hour.    -   5. Same as General Procedure.    -   6. Same as General Procedure.    -   7. The blocking solution is removed by decanting and the wells        are washed once with PBS.    -    Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is added        (100 μl/well) and the plate    -    is incubated for 90 minutes at room temperature on a plate        shaker.    -   8. Same as General Procedure.    -   9. Same as General Procedure.    -   10. Same as General Procedure.        HUV-EC-C Assay

This assay is used to measure a compound's activity against PDGF-R,FGF-R, VEGF, aFGF or Flk-1/KDR, all of which are naturally expressed byHUV-EC cells.

Day 0

1. Wash and trypsinize HUV-EC-C cells (human umbilical vein endothelialcells, (American Type Culture Collection, catalogue no. 1730 CRL). Washwith Dulbecco's phosphate-buffered saline (D-PBS, obtained from GibcoBRL, catalogue no. 14190-029) 2 times at about 1 ml/10 cm² of tissueculture flask. Trypsinize with 0.05% trypsin-EDTA in non-enzymatic celldissociation solution (Sigma Chemical Company, catalogue no. C-1544).The 0.05% trypsin is made by diluting 0.25% trypsin/1 mM EDTA (Gibco,catalogue no. 25200-049) in the cell dissociation solution. Trypsinizewith about 1 ml/25-30 cm² of tissue culture flask for about 5 minutes at37° C. After cells have detached from the flask, add an equal volume ofassay medium and transfer to a 50 ml sterile centrifuge tube (FisherScientific, catalogue no. 05-539-6).

2. Wash the cells with about 35 ml assay medium in the 50 ml sterilecentrifuge tube by adding the assay medium, centrifuge for 10 minutes atapproximately 200× g, aspirate the supernatant, and resuspend with 35 mlD-PBS. Repeat the wash two more times with D-PBS, resuspend the cells inabout 1 ml assay medium/15 cm² of tissue culture flask. Assay mediumconsists of F12K medium (Gibco BRL, catalogue no. 21127-014) and 0.5%heat-inactivated fetal bovine serum. Count the cells with a CoulterCounter® (Coulter Electronics, Inc.) and add assay medium to the cellsto obtain a concentration of 0.8-1.0×10⁵ cells/ml.

3. Add cells to 96-well flat-bottom plates at 100 μl/well or 0.8-1.0×10⁴cells/well, incubate ˜24 h at 37° C., 5% CO₂.

Day 1

1. Make up two-fold test compound titrations in separate 96-well plates,generally 50 μM on down to 0 μM. Use the same assay medium as mentionedin day 0, step 2 above. Titrations are made by adding 90 μl/well of testcompound at 200 μM (4× the final well concentration) to the top well ofa particular plate column. Since the stock test compound is usually 20mM in DMSO, the 200 μM drug concentration contains 2% DMSO.

A diluent made up to 2% DMSO in assay medium (F12K+0.5% fetal bovineserum) is used as diluent for the test compound titrations in order todilute the test compound but keep the DMSO concentration constant. Addthis diluent to the remaining wells in the column at 60 μl/well. Take 60μl from the 120 μl of 200 μM test compound dilution in the top well ofthe column and mix with the 60 μl in the second well of the column. Take60 μl from this well and mix with the 60 μl in the third well of thecolumn, and so on until two-fold titrations are completed. When thenext-to-the-last well is mixed, take 60 μl of the 120 μl in this welland discard it. Leave the last well with 60 μl of DMSO/media diluent asa non-test compound-containing control. Make 9 columns of titrated testcompound, enough for triplicate wells each for: (1) VEGF (obtained fromPepro Tech Inc., catalogue no. 100-200, (2) endothelial cell growthfactor (ECGF) (also known as acidic fibroblast growth factor, or aFGF)(obtained from Boehringer Mannheim Biochemica, catalogue no. 1439 600),or, (3) human PDGF B/B (1276-956, Boehringer Mannheim, Germany) andassay media control. ECGF comes as a preparation with sodium heparin.

2. Transfer 50 μl/well of the test compound dilutions to the 96-wellassay plates containing the 0.8-1.0×10⁴ cells/100 μl/well of theHUV-EC-C cells from day 0 and incubate ˜2 h at 37° C., 5% CO₂.

3. In triplicate, add 50 μl/well of 80 μg/ml VEGF, 20 ng/ml ECGF, ormedia control to each test compound condition. As with the testcompounds, the growth factor concentrations are 4× the desired finalconcentration. Use the assay media from day 0 step 2 to make theconcentrations of growth factors. Incubate approximately 24 hours at 37°C., 5% CO₂. Each well will have 50 μl test compound dilution, 50 μlgrowth factor or media, and 100 μl cells, which calculates to 200μl/well total. Thus the 4× concentrations of test compound and growthfactors become 1× once everything has been added to the wells.

Day 2

1. Add ³H-thymidine (Amersham, catalogue no. TRK-686) at 1 μCi/well (10μl/well of 100 μCi/ml solution made up in RPMI media +10%heat-inactivated fetal bovine serum) and incubate ˜24 h at 37° C., 5%CO₂. RPMI is obtained from Gibco BRL, catalogue no. 11875-051.

Day 3

1. Freeze plates overnight at −20° C.

Day 4

Thaw plates and harvest with a 96-well plate harvester (Tomtec Harvester96®) onto filter mats (Wallac, catalogue no. 1205-401), read counts on aWallac Betaplate™ liquid scintillation counter.

Bioassays which have been or can be used to evaluate compounds aredescribed in detail below. Compounds 1-9 were tested and found active inflkGST, FGFR1 and PDGF assays.

In Vivo Animal Models

Xenograft Animal Models

The ability of human tumors to grow as xenografts in athymic mice (e.g.,Balb/c, nu/nu) provides a useful in vivo model for studying thebiological response to therapies for human tumors. Since the firstsuccessful xenotransplantation of human tumors into athymic mice,(Rygaard and Povlsen, 1969, Acta Pathol. Microbial. Scand. 77:758-760),many different human tumor cell lines (e.g., mammary, lung,genitourinary, gastro-intestinal, head and neck, glioblastoma, bone, andmalignant melanomas) have been transplanted and successfully grown innude mice. The following assays may be used to determine the level ofactivity, specificity and effect of the different compounds of thepresent invention. Three general types of assays are useful forevaluating compounds: cellular/catalytic, cellular/biological and invivo. The object of the cellular/catalytic assays is to determine theeffect of a compound on the ability of a TK to phosphorylate

tyrosines on a known substrate in a cell. The object of thecellular/biological assays is to determine the effect of a compound onthe biological response stimulated by a TK in a cell. The object of thein vivo assays is to determine the effect of a compound in an animalmodel of a particular disorder such as cancer.

Suitable cell lines for subcutaneous xenograft experiments include C6cells (glioma, ATCC # CCL 107), A375 cells (melanoma, ATCC # CRL 1619),A431 cells (epidermoid carcinoma, ATCC # CRL 1555), Calu 6 cells (lung,ATCC # HTB 56), PC3 cells (prostate, ATCC # CRL 1435), SKOV3TP5 cellsand NIH 3T3 fibroblasts genetically engineered to overexpress EGFR,PDGFR, IGF-1R or any other test kinase. The following protocol can beused to perform xenograft experiments:

Female athymic mice (BALB/c, nu/nu) are obtained from SimonsenLaboratories (Gilroy, Calif.). All animals are maintained underclean-room conditions in Micro-isolator cages with Alpha-dri bedding.They receive sterile rodent chow and water ad libitum.

Cell lines are grown in appropriate medium (for example, MEM, DMEM,Ham's F10, or Ham's F12 plus 5%-10% fetal bovine serum (FBS) and 2 mMglutamine (GLN)). All cell culture media, glutamine, and fetal bovineserum are purchased from Gibco Life Technologies (Grand Island, N.Y.)unless otherwise specified. All cells are grown in a humid atmosphere of90-95% air and 5-10% CO₂ at 37° C. All cell lines are routinelysubcultured twice a week and are negative for mycoplasma as determinedby the Mycotect method (Gibco).

Cells are harvested at or near confluency with 0.05% Trypsin-EDTA andpelleted at 450×g for 10 min. Pellets are resuspended in sterile PBS ormedia (without FBS) to a particular concentration and the cells areimplanted into the hindflank of the mice (8-10 mice per group, 2-10×10⁶cells/animal). Tumor growth is measured over 3 to 6 weeks using veniercalipers. Tumor volumes are calculated as a product oflength×width×height unless otherwise indicated. P values are calculatedusing the Students t-test. Test compounds in 50-100 μL excipient (DMSO,or VPD:D5W) can be delivered by IP injection at different concentrationsgenerally starting at day one after implantation.

Tumor Invasion Model

The following tumor invasion model has been developed and may be usedfor the evaluation of therapeutic value and efficacy of the compoundsidentified to selectively inhibit KDR/FLK-1 receptor.

Procedure

8 week old nude mice (female) (Simonsen Inc.) are used as experimentalanimals. Implantation of tumor cells can be performed in a laminar flowhood. For anesthesia, Xylazine/Ketamine Cocktail (100 mg/kg ketamine and5 mg/kg Xylazine) are administered intraperitoneally. A midline incisionis done to expose the abdominal cavity (approximately 1.5 cm in length)to inject 10⁷ tumor cells in a volume of 100 μl medium. The cells areinjected either into the duodenal lobe of the pancreas or under theserosa of the colon. The peritoneum and muscles are closed with a 6-0silk continuous suture and the skin is closed by using wound clips.Animals are observed daily.

Analysis

After 2-6 weeks, depending on gross observations of the animals, themice are sacrificed, and the local tumor metastases to various organs(lung, liver, brain, stomach, spleen, heart, muscle) are excised andanalyzed (measurement of tumor size, grade of invasion, immunochemistry,in situ hybridization determination, etc.).

C-Kit Assay

This assay is used to detect the level of c-kit tyrosinephosphorylation.

MO7E (human acute myeloid leukemia) cells are serum starved overnight in0.1% serum. Cells are pre-treated with the compound (concurrent withserum starvation), prior to ligand stimulation. Cells are stimulatedwith 250 ng/ml rh-SCF for 15 minutes. Following stimulation, cells werelysed and immunoprecipitated with an anti-c-kit antibody.Phosphotyrosine and protein levels were determined by Western blotting.

MTT Proliferation Assay

MO7E cells are serum starved and pre-treated with compound as describedfor the phosphorylation experiments. Cells areplated @ 4×10⁵ cells/wellin a 96 well dish, in 100 μl RPMI+10% serum. rh-SCF (100 ng/mL) is addedand the plate is incubated for 48 hours. After 48 hours, 10 μl of 5mg/ml MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide) is added and allowed to incubate for 4 hours. Acid isopropanol(100 μl of 0.04N HCl in isopropanol) is added and the optical densitywas measured at a wavelength of 550 nm.

Apoptosis Assay

MO7E cells are incubated +/−SCF and +/−compound in 10% FBS withrh-GM-CSF (10 ng/mL) and rh-IL-3 (10 ng/mL). Samples are assayed at 24and 48 hours. To measure activated caspase-3, samples are washed withPBS and permeabilized with ice-cold 70% ethanol. The cells are thenstained with PE-conjugated polyclonal rabbit anti-active caspase-3 andanalyzed by FACS. To measure cleaved PARP, samples are lysed andanalyzed by western blotting with an anti-PARP antibody.

Additional Assays

Additional assays which may be used to evaluate the compounds of thisinvention include, without limitation, a bio-flk-1 assay, an EGFreceptor-HER2 chimeric receptor assay in whole cells, a bio-src assay, abio-Ick assay and an assay measuring the phosphorylation function ofraf. The protocols for each of these assays may be found in U.S.application Ser. No. 09/099,842, which is incorporated by reference,including any drawings, herein.

Measurement of Cell Toxicity

Therapeutic compounds should be more potent in inhibiting receptortyrosine kinase activity than in exerting a cytotoxic effect. A measureof the effectiveness and cell toxicity of a compound can be obtained bydetermining the therapeutic index, i.e., IC₅₀/LD₅₀. IC₅₀, the doserequired to achieve 50% inhibition, can be measured using standardtechniques such as those described herein. LD₅₀, the dosage whichresults in 50% toxicity, can also be measured by standard techniques aswell (Mossman, 1983, J. Immunol. Methods, 65:55-63), by measuring theamount of LDH released (Korzeniewski and Callewaert, 1983, J. Immunol.Methods, 64:313, Decker and Lohmann-Matthes, 1988, J. Immunol. Methods,115:61), or by measuring the lethal dose in animal models. Compoundswith a large therapeutic index are preferred. The therapeutic indexshould be greater than 2, preferably at least 10, more preferably atleast 50.

One skilled in the art would also readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent herein. Themolecular complexes and the methods, procedures, treatments, molecules,specific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group. For example, if X isdescribed as selected from the group consisting of bromine, chlorine,and iodine, claims for X being bromine and claims for X being bromineand chlorine are fully described.

1. A method of making a compound of the formula

or a pharmaceutically acceptable salt thereof, comprising reacting acompound of the formula

with a compound of the formula

and a compound of the formula

in the presence of a suitable base and in a suitable solvent.
 2. Themethod of claim 1, wherein the suitable base is triethyl amine.
 3. Themethod of claim 1, wherein the suitable solvent is tetrahydrofuran.